Labeling and detection of nucleic acids

ABSTRACT

Provided in certain embodiments are new methods for forming azido modified nucleic acid conjugates of reporter molecules, carrier molecules or solid support. In other embodiments are provided methods for enzymatically labeling nucleic acids with an azide group.

CROSS REFERENCE TO RELATED APPLICATION(S)

This application is a Continuation-in-Part of U.S. Ser. No. 11/674,140,filed Feb. 12, 2007, which claims priority to U.S. ProvisionalApplication No. 60/772,221, filed Feb. 10, 2006 and U.S. ProvisionalApplication No. 60/804,640, filed Jun. 13, 2006, the contents of whichare incorporated by reference as if set forth fully herein.

FIELD OF THE INVENTION

The invention generally relates to methods of labeling nucleic acidpolymers and their use.

BACKGROUND INFORMATION

Conventional methods for labeling nucleotides are straightforward, buthave significant drawbacks. With direct fluorophore labeling, the bulkydye molecule on the nucleotide makes it difficult for the enzyme toincorporate nucleotides into DNA or RNA strands. Additionally, protocolsoptimized for one fluorophore may not be optimal for another, chemicallydifferent fluorophore.

Various methods have been used to generate labeled probes forhybridization to Southern blots and microarrays, for example, 5′ and 3′end labeling with ³²P. Additionally, nick translation uses DNAse I togenerate single stranded nicks in the nucleic acid starting material andDNA polymerase to fill in the nicks. A labeled deoxynucleotide (forexample dUTP-digoxigenin or dUTP fluorescein) is included in thereaction mixture, along with the other unlabeled deoxynucleotides. Whilethese methods generate labeled probes, they do not provide a method ofamplifying the starting material.

Polymerase chain reaction (PCR) in the presence of a mixture ofnucleotides (for example dATP, dCTP, dGTP, dTTP, and modifieddUTP-digoxigenin or fluorescein) can be used to synthesize copies of atemplate strand. These amplicons can be used as probes for hybridizationassays. The mixture must contain unmodified dTTP in addition to modifieddUTP in order for the reaction to take place.

PCR uses a double stranded DNA template as starting material. Thistemplate can be made from RNA by reverse transcription and subsequentlylabeled by PCR incorporation of labeled nucleotides. While this methoddoes result in an amplification of the starting material, thesubstitution of the deoxynucleotide fluorescent analogue is less than100% and the specific activity may be variable, depending on the label.

Accordingly, one object of the present invention is to provide animproved method for nucleotide labeling that circumvents problemsassociated with conventional methods. Preferably, the methods will beamenable to a variety of uses including generating FISH probes,generating probes for Southern blots, generating probes for Northernblots, colorimetric in situ hybridization probes (CISH), in situ PCR,isothermal amplification in situ, DNA fingerprinting, and SNP detection.

SUMMARY OF THE INVENTION

One aspect of the invention provides a method of forming a nucleic acidconjugate, wherein the method comprises:

-   -   incorporating an azide modified nucleotide into the nucleic acid        polymer by contacting the azide modified nucleotide nucleotide        with at least one other nucleotide in the presence of a DNA        amplification enzyme to form an azide modified nucleic acid        polymer; and    -   contacting the azide modified nucleic acid polymer with a        reporter molecule, carrier molecule or solid support that        comprises an activated or terminal alkyne or phosphine moiety to        form a nucleic acid polymer-reporter molecule, carrier molecule,        solid support conjugate.

Another aspect of the invention provides method of forming a nucleicacid conjugate, wherein the method comprises:

-   -   incorporating a terminal alkyne modified nucleotide into the        nucleic acid polymer by contacting the terminal alkyne modified        nucleotide nucleotide with at least one other nucleotide in the        presence of a DNA amplification enzyme to form a terminal alkyne        modified nucleic acid polymer; and    -   contacting the terminal alkyne modified nucleic acid polymer        with a reporter molecule, carrier molecule or solid support that        comprises an azido moiety to form a nucleic acid        polymer-reporter molecule, carrier molecule, solid support        conjugate.

Another aspect of the invention provides a method of forming a nucleicacid conjugate, wherein the method comprises:

-   -   incorporating a phosphine modified nucleotide into the nucleic        acid polymer by contacting the phosphine modified nucleotide        nucleotide with at least one other nucleotide in the presence of        a DNA amplification enzyme to form a phosphine modified nucleic        acid polymer; and    -   contacting the phosphine modified nucleic acid polymer with a        reporter molecule, carrier molecule or solid support that        comprises an azido moiety to form a nucleic acid        polymer-reporter molecule, carrier molecule, solid support        conjugate.

Another aspect of the invention provides a method for making an azido,alkyne or phosphine modified nucleic acid polymer, wherein the methodcomprises:

-   -   incubating at least one azido, alkyne or phosphine modified        nucleotide in the presence of a nucleic acid amplification        enzyme to form an azido, alkyne or phosphine modified nucleic        acid polymer.

In another embodiment, the nucleic acid enzyme is a DNA polymerase.

In another embodiment, the nucleic acid enzyme is a RNA polymerase.

In another embodiment, the melting temperature of the azido, alkyne orphosphine modified nucleic acid polymer is increased.

In another embodiment, the reporter molecule is a xanthene, cyanine,coumarin, borapolyazaindacene or pyrene dye. In another embodiment, thereporter molecule is an enzyme substrate or hapten.

In another embodiment, the carrier molecule is an amino acid, a peptide,a protein, a polysaccharide, a nucleotide, a nucleoside, anoligonucleotide, a nucleic acid, a hapten, a psoralen, a drug, ahormone, a lipid, a lipid assembly, a synthetic polymer, a polymericmicroparticle, a biological cell or a virus. In another embodiment, thecarrier molecule comprises an antibody or fragment thereof, an avidin orstreptavidin, a biotin, a blood component protein, a dextran, an enzyme,an enzyme inhibitor, a hormone, an IgG binding protein, a fluorescentprotein, a growth factor, a lectin, a lipopolysaccharide, amicroorganism, a metal binding protein, a metal chelating moiety, anon-biological microparticle, a peptide toxin, aphosphotidylserine-binding protein, a structural protein, asmall-molecule drug, or a tyramide.

In another embodiment, the solid support is a microfluidic chip, asilicon chip, a microscope slide, a microplate well, silica gels,polymeric membranes, particles, derivatized plastic films, glass beads,cotton, plastic beads, alumina gels, polysaccharides, polyvinylchloride,polypropylene, polyethylene, nylon, latex bead, magnetic bead,paramagnetic bead, or superparamagnetic bead.

In another embodiment, the solid support is Sepharose, poly(acrylate),polystyrene, poly(acrylamide), polyol, agarose, agar, cellulose,dextran, starch, FICOLL, heparin, glycogen, amylopectin, mannan, inulin,nitrocellulose, diazocellulose or starch.

Another aspect of the invention provides a method of detecting an azidomodified nucleic acid polymer, comprising:

-   -   forming an azide-alkyne cycloaddition reaction mixture        comprising: a reporter molecule that comprises a terminal alkyne        moiety:    -   an azido modified nucleic acid polymer;    -   incubating the azide-alkyne cycloaddition reaction mixture for a        sufficient amount of time to form a nucleic acid        polymer-reporter molecule conjugate;    -   separating the nucleic acid polymer-reporter conjugate by size        and/or weight of the nucleic acid polymer-reporter-reporter        molecule conjugate to form a separated nucleic acid        polymer-reporter-reporter molecule conjugate;    -   illuminating the separated nucleic acid        polymer-reporter-reporter molecule conjugate with an appropriate        wavelength to form an illuminated nucleic acid        polymer-reporter-reporter molecule conjugate;    -   observing the illuminated nucleic acid polymer-reporter-reporter        molecule conjugate wherein the nucleic acid polymer is detected.

In a more particular embodiment the forming step further comprises

-   -   a. copper ions;    -   b. at least one reducing agent; and    -   c. a copper chelator.

In another embodiment, the reporter molecule is xanthene, cyanine,coumarin, borapolyazaindacene or pyrene dye. In another embodiment, thereporter molecule is an enzyme substrate, fluorescent protein or hapten.

In another embodiment, the copper chelator is a copper (I) chelator. Inanother embodiment, the copper chelator isN,N,N′,N′-tetrakis(2-pyridylmethyl)ethylenediamine (TPEN), EDTA,neocuproine, N-(2-acetamido)iminodiacetic acid (ADA),pyridine-2,6-dicarboxylic acid (PDA), S-carboxymethyl-L-cysteine (SCMC),1,10 phenanthroline, or a derivative thereof, trientine, glutathione,histadine, polyhistadine or tetra-ehylenepolyamine (TEPA). In anotherembodiment, the copper chelator is 1,10 phenanthroline,bathophenanthroline disulfonic acid (4,7odiphenyl-1,10-phenanthrolinedisulfonic acid) or bathocuproine disulfonic acid(2,9-dimethyl-4,7-diphenyl-1,10-phenanthroline disulfonate).

In another embodiment, the reducing agent is acorbate,Tris(2-Carboxyethyl) Phosphine (TCEP), TCP (2,4,6-trichlorophenol),NADH, NADPH, thiosulfate, 2-mercaptoethanol, dithiothreotol,glutathione, cysteine, metallic copper, quinone, hydroquinone, vitaminK₁, Fe²⁺, Co²⁺, or an applied electric potential. In another embodiment,the reducing agent is ascorbate.

In another embodiment, the separating step comprises chromatography orelectrophoresis. In another embodiment, the chromatography comprises oneor more of FPLC, HPLC, liquid chromatograpy (LC), size exclusionchromatography, ion exchange chromatography, or affinity chromatography.

In another embodiment, electrophoresis comprises gel electrophoresis, 1dimensional (1D) gel electrophoresis, 2 dimensional (2D) gelelectrophoresis, native gel electrophoresis, denaturing gelelectrophoresis, isoelectric focusing, or capillary electrophoresis.

Another aspect of the invention provides an azide-alkyne cycloadditionreaction mixture comprising:

-   -   a reporter molecule that comprises a terminal alkyne moiety:    -   an azido modified nucleic acid;    -   copper ions;    -   at least one reducing agent; and    -   a copper chelator.

Another aspect of the invention provides a method for detectingimmobilized azido modified nucleic acids, wherein the method comprises:

-   -   immobilizing the azido modified nucleic acids on a solid or        semi-solid matrix to form an immobilized azido modified nucleic        acid;    -   contacting the immobilized azido modified nucleic acid with a        reporter molecule that contains an azide reactive group to form        a contacted azido modified nucleic acid;    -   incubating the contacted azido modified nucleic acid for a        sufficient amount of time to form a reporter molecule-nucleic        acid conjugate;    -   illuminating the reporter molecule-nucleic acid conjugate with        an appropriate wavelength to form an illuminated reporter        molecule-nucleic acid conjugate;    -   observing the illuminated reporter molecule-nucleic acid        conjugate whereby the immobilized azido modified nucleic acid is        detected.

Another aspect of the invention provides a method for detectingimmobilized alkyne modified nucleic acids, wherein the method comprises:

-   -   immobilizing the alkyne modified nucleic acids on a solid or        semi-solid matrix to form an immobilized alkyne modified nucleic        acid;    -   contacting the immobilized alkyne modified nucleic acid with a        reporter molecule that contains an azido group to form a        contacted alkyne modified nucleic acid;    -   incubating the contacted alkyne modified nucleic acid for a        sufficient amount of time to form a reporter molecule-nucleic        acid conjugate;    -   illuminating the reporter molecule-nucleic acid conjugate with        an appropriate wavelength to form an illuminated reporter        molecule-nucleic acid conjugate;    -   observing the illuminated reporter molecule-nucleic acid        conjugate whereby the immobilized alkyne modified nucleic acid        is detected.

In another embodiment, the solid or semi-solid support is a slide, anarray, an agarose gel, a polyacrylamide gel, a hydrogel, a polymericparticle or glass.

Another aspect of the invention provides a kit comprising:

-   -   Azide-dATP;    -   a telomerase enzyme;    -   an azide reactive reporter molecule, carrier molecule or solid        support.

Another aspect of the invention provides a kit for labeling a nucleicacid polymer comprising:

-   -   at least one nucleotide analogue that comprises an azide, alkyne        or phosphine moiety; and    -   a reporter molecule, carrier molecule or solid support        comprising an azide, alkyne or phosphine moiety.

A more particular embodiment thereof further comprises a nucleic acidamplification enzyme.

Another aspect of the invention provides a method of measuringTelomerase Enzyme Activity, comprising steps of:

-   -   a) contacting a cell with an effective amount of a dNTP        nucleotide that comprises an azide group and a Telomerase enzyme        such that the dNTP nucleotide is incorporated into at least one        nucleic acid polymer;    -   b) contacting the nucleic acid polymer with a reporter molecule        comprising an alkyne or phosphine moiety to form a azido        modified nucleic acid polymer reporter molecule conjugate;    -   c) separating the azido modified nucleic acid polymer reporter        molecule conjugate from nucleic acid polymers that do not        comprise a reporter molecule, and    -   d) illuminating the azido modified nucleic acid polymer reporter        molecule conjugate to determine Telomerase activity.

Another aspect of the invention provides a method of measuringTelomerase Enzyme Activity, comprising steps of:

-   -   a) contacting a cell with an effective amount of a dNTP        nucleotide that comprises an alkyne or phosphine group and a        Telomerase enzyme such that the dNTP nucleotide is incorporated        into at least one nucleic acid polymer;    -   b) contacting the nucleic acid polymer with a reporter molecule        comprising an azido moiety to form an alkyne or phosphine        modified nucleic acid polymer reporter molecule conjugate;    -   c) separating the alkyne or phosphine modified nucleic acid        polymer reporter molecule conjugate from nucleic acid polymers        that do not comprise a reporter molecule, and    -   d) illuminating the alkyne or phosphine modified nucleic acid        polymer reporter molecule conjugate to determine Telomerase        activity.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. (1): Shows in gel detection Telomerase incorporation of N₃-dATP.

FIG. (2): Shows in gel detection and dose-dependence of Telomeraseincorporation of N₃-dATP and not N₃-dAUP.

FIG. (3): Shows in gel detection and dose-dependence of Telomeraseincorporation of N₃-dATP and labeled with alkyne-TAMRA using “click”chemistry.

FIG. (4): Shows in gel detection of Telomerase incorporation ofethynyl-dAUP and labeled with azide-TAMRA using “click” chemistry.

FIG. (5): Shows in gel detection of Telomerase incorporation ofethynyl-dAUP and labeled with azide-TAMRA using “click” chemistry.

FIG. (6): Shows in gel detection of incorporation of ethynyl-dAUP usinglinear amplification and various polymerases. The ethynyl-dAUP waslabeled with azide-TAMRA using “click” chemistry.

FIG. (7): Shows that the presence of a copper chelator (BCS) maintainsTelomerase laddering.

FIG. (8): Is a schematic diagram showing amplification using helicaseenzyme.

FIG. (9): Is a schematic diagram showing Strand DisplacementAmplification (SDA)

FIG. (10): Illustration of the position of the primers used in Example13, giving a predicted amplicon size of 293 bp.

FIG. (11): Is a linear amplification plot showing that the onset ofexponential amplification or threshold cycle (CT) for the click modifieddUTP mix is very similar to that shown for the unmodified dUTP mix. Theaverage CT for click dUTP is 9.78 vs 10.97 for unmodified dUTP.

FIG. (12): Shows dissociation curves for the reactions in Example 13,showing the change in fluorescence as a function of temperature. Clickmodified dUTP mix melts at 93 C, vs unmodified dUTP mix at 88 C.

FIG. (13): Gel analysis of real-time PCR products scanned for TAMRA(left) or scanned after labeling with SYBR GOLD (right).

FIG. (14). shows images of reaction tubes for TAMRA.

FIG. (15): shows a dot blot of reaction products scanned for TAMRA.

FIG. (16): shows the experimental setup for Example 15.

DETAILED DESCRIPTION OF THE INVENTION Definitions and Abbreviations

Before describing the present invention in detail, it is to beunderstood that this invention is not limited to specific compositionsor process steps, as such may vary. It must be noted that, as used inthis specification and the appended claims, the singular form “a”, “an”and “the” include plural referents unless the context clearly dictatesotherwise. Thus, for example, reference to “a ligand” includes aplurality of ligands and reference to “a nucleic acid” includes aplurality of nucleic acids and the like.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention is related. The following terms andabbreviations (Table 1) are defined for purposes of the invention asdescribed herein.

TABLE 1 List of Abbreviations Abbreviation Term. E-dNTP ethynyldeoxynucleoside triphosphate E-ATP ethynyl deoxyadenosine triphosphateE-GTP ethynyl deoxyguanosine triphosphate E-CTP ethynyl deoxycytidinetriphosphate E-TTP ethynyl deoxythymidine triphosphate

Certain compounds of the present invention can exist in unsolvated formsas well as solvated forms, including hydrated forms. In general, thesolvated forms are equivalent to unsolvated forms and are encompassedwithin the scope of the present invention. Certain compounds of thepresent invention may exist in multiple crystalline or amorphous forms.In general, all physical forms are equivalent for the uses contemplatedby the present invention and are intended to be within the scope of thepresent invention.

Certain compounds of the present invention possess asymmetric carbonatoms (optical centers) or double bonds; the racemates, diastereomers,geometric isomers and individual isomers are encompassed within thescope of the present invention.

The compounds described herein may be prepared as a single isomer (e.g.,enantiomer, cis-trans, positional, diastereomer) or as a mixture ofisomers. In a preferred embodiment, the compounds are prepared assubstantially a single isomer. Methods of preparing substantiallyisomerically pure compounds are known in the art. For example,enantiomerically enriched mixtures and pure enantiomeric compounds canbe prepared by using synthetic intermediates that are enantiomericallypure in combination with reactions that either leave the stereochemistryat a chiral center unchanged or result in its complete inversion.Alternatively, the final product or intermediates along the syntheticroute can be resolved into a single stereoisomer. Techniques forinverting or leaving unchanged a particular stereocenter, and those forresolving mixtures of stereoisomers are well known in the art and it iswell within the ability of one of skill in the art to choose andappropriate method for a particular situation. See, generally, Furnisset al. (eds.), VOGEL'S ENCYCLOPEDIA OF PRACTICAL ORGANIC CHEMISTRY5^(TH) ED., Longman Scientific and Technical Ltd., Essex, 1991, pp.809-816; and Heller, Acc. Chem. Res. 23: 128 (1990).

The compounds disclosed herein may also contain unnatural proportions ofatomic isotopes at one or more of the atoms that constitute suchcompounds. For example, the compounds may be radiolabeled withradioactive isotopes, such as for example tritium (³H), iodine-125(¹²⁵I) or carbon-14 (¹⁴C). All isotopic variations of the compounds ofthe present invention, whether radioactive or not, are intended to beencompassed within the scope of the present invention.

Where a disclosed compound includes a conjugated ring system, resonancestabilization may permit a formal electronic charge to be distributedover the entire molecule. While a particular charge may be depicted aslocalized on a particular ring system, or a particular heteroatom, it iscommonly understood that a comparable resonance structure can be drawnin which the charge may be formally localized on an alternative portionof the compound.

Selected compounds having a formal electronic charge may be shownwithout an appropriate biologically compatible counterion. Such acounterion serves to balance the positive or negative charge present onthe compound. As used herein, a substance that is biologicallycompatible is not toxic as used, and does not have a substantiallydeleterious effect on biomolecules. Examples of negatively chargedcounterions include, among others, chloride, bromide, iodide, sulfate,alkanesulfonate, arylsulfonate, phosphate, perchlorate,tetrafluoroborate, tetraarylboride, nitrate and anions of aromatic oraliphatic carboxylic acids. Preferred counterions may include chloride,iodide, perchlorate and various sulfonates. Examples of positivelycharged counterions include, among others, alkali metal, or alkalineearth metal ions, ammonium, or alkylammonium ions.

Where substituent groups are specified by their conventional chemicalformulae, written from left to right, they equally encompass thechemically identical substituents, which would result from writing thestructure from right to left, e.g., —CH₂O— is intended to also recite—OCH₂—.

The term “acyl” or “alkanoyl” by itself or in combination with anotherterm, means, unless otherwise stated, a stable straight or branchedchain, or cyclic hydrocarbon radical, or combinations thereof,consisting of the stated number of carbon atoms and an acyl radical onat least one terminus of the alkane radical. The “acyl radical” is thegroup derived from a carboxylic acid by removing the —OH moietytherefrom.

The term “alkyl,” by itself or as part of another substituent means,unless otherwise stated, a straight or branched chain, or cyclichydrocarbon radical, or combination thereof, which may be fullysaturated, mono- or polyunsaturated and can include divalent(“alkylene”) and multivalent radicals, having the number of carbon atomsdesignated (i.e. C₁-C₁₀ means one to ten carbons). Examples of saturatedhydrocarbon radicals include, but are not limited to, groups such asmethyl, ethyl, n-propyl, isopropyl, n-butyl, t-butyl, isobutyl,sec-butyl, cyclohexyl, (cyclohexyl)methyl, cyclopropylmethyl, homologsand isomers of, for example, n-pentyl, n-hexyl, n-heptyl, n-octyl, andthe like. An unsaturated alkyl group is one having one or more doublebonds or triple bonds. Examples of unsaturated alkyl groups include, butare not limited to, vinyl, 2-propenyl, crotyl, 2-isopentenyl,2-(butadienyl), 2,4-pentadienyl, 3-(1,4-pentadienyl), ethynyl, 1- and3-propynyl, 3-butynyl, and the higher homologs and isomers. The term“alkyl,” unless otherwise noted, is also meant to include thosederivatives of alkyl defined in more detail below, such as“heteroalkyl.” Alkyl groups that are limited to hydrocarbon groups aretermed “homoalkyl”.

Exemplary alkyl groups of use in the present invention contain betweenabout one and about twenty five carbon atoms (e.g. methyl, ethyl and thelike). Straight, branched or cyclic hydrocarbon chains having eight orfewer carbon atoms will also be referred to herein as “lower alkyl”. Inaddition, the term “alkyl” as used herein further includes one or moresubstitutions at one or more carbon atoms of the hydrocarbon chainfragment.

The term “amino” or “amine group” refers to the group —NR′R″ (or NRR′R″)where R, R′ and R″ are independently selected from the group consistingof hydrogen, alkyl, substituted alkyl, aryl, substituted aryl, arylalkyl, substituted aryl alkyl, heteroaryl, and substituted heteroaryl. Asubstituted amine being an amine group wherein R′ or R″ is other thanhydrogen. In a primary amino group, both R′ and R″ are hydrogen, whereasin a secondary amino group, either, but not both, R′ or R″ is hydrogen.In addition, the terms “amine” and “amino” can include protonated andquaternized versions of nitrogen, comprising the group —NRR′R″ and itsbiologically compatible anionic counterions.

The term “aryl” as used herein refers to cyclic aromatic carbon chainhaving twenty or fewer carbon atoms, e.g., phenyl, naphthyl, biphenyl,and anthracenyl. One or more carbon atoms of the aryl group may also besubstituted with, e.g., alkyl; aryl; heteroaryl; a halogen; nitro;cyano; hydroxyl, alkoxyl or aryloxyl; thio or mercapto, alkyl-, orarylthio; amino, alkylamino, arylamino, dialkyl-, diaryl-, orarylalkylamino; aminocarbonyl, alkylaminocarbonyl, arylaminocarbonyl,dialkylaminocarbonyl, diarylaminocarbonyl, or arylalkylaminocarbonyl;carboxyl, or alkyl- or aryloxycarbonyl; aldehyde; aryl- oralkylcarbonyl; iminyl, or aryl- or alkyliminyl; sulfo; alkyl- oralkylcarbonyl; iminyl, or aryl- or alkyliminyl; sulfo; alkyl- orarylsufonyl; hydroximinyl, or aryl- or alkoximinyl. In addition, two ormore alkyl or heteroalkyl substituents of an aryl group may be combinedto form fused aryl-alkyl or aryl-heteroalkyl ring systems (e.g.,tetrahydronaphthyl). Substituents including heterocyclic groups (e.g.,heteroaryloxy, and heteroaralkylthio) are defined by analogy to theabove-described terms.

The terms “alkoxy,” “alkylamino” and “alkylthio” (or thioalkoxy) areused in their conventional sense, and refer to those alkyl groupsattached to the remainder of the molecule via an oxygen atom, an aminogroup, or a sulfur atom, respectively.

The term “heteroalkyl,” by itself or in combination with another term,means, unless otherwise stated, a straight or branched chain, or cycliccarbon-containing radical, or combinations thereof, consisting of thestated number of carbon atoms and at least one heteroatom selected fromthe group consisting of O, N, Si, P, S, and Se and wherein the nitrogen,phosphorous, sulfur, and selenium atoms are optionally oxidized, and thenitrogen heteroatom is optionally be quaternized. The heteroatom(s) O,N, P, S, Si, and Se may be placed at any interior position of theheteroalkyl group or at the position at which the alkyl group isattached to the remainder of the molecule. Examples include, but are notlimited to, —CH₂—CH₂—O—CH₃, —CH₂—CH₂—NH—CH₃, —CH₂—CH₂—N(CH₃)—CH₃,—CH₂—S—CH₂—CH₃, —CH₂—CH₂, —S(O)—CH₃, —CH₂—CH₂—S(O)₂—CH₃, —CH═CH—O—CH₃,—Si(CH₃)₃, —CH₂—CH═N—OCH₃, and —CH═CH—N(CH₃)—CH₃. Up to two heteroatomsmay be consecutive, such as, for example, —CH₂—NH—OCH₃ and—CH₂—O—Si(CH₃)₃. Similarly, the term “heteroalkylene” by itself or aspart of another substituent means a divalent radical derived fromheteroalkyl, as exemplified, but not limited by, —CH₂—CH₂—S—CH₂—CH₂— and—CH₂—S—CH₂—CH₂—NH—CH₂—. For heteroalkylene groups, heteroatoms can alsooccupy either or both of the chain termini (e.g., alkyleneoxy,alkylenedioxy, alkyleneamino, alkylenediamino, and the like). Stillfurther, for alkylene and heteroalkylene linking groups, no orientationof the linking group is implied by the direction in which the formula ofthe linking group is written. For example, the formula —C(O)₂R′—represents both —C(O)₂R′— and —R′C(O)₂—.

The terms “cycloalkyl” and “heterocycloalkyl”, by themselves or incombination with other terms, represent, unless otherwise stated, cyclicversions of “alkyl” and “heteroalkyl”, respectively. Additionally, forheterocycloalkyl, a heteroatom can occupy the position at which theheterocycle is attached to the remainder of the molecule. Examples ofcycloalkyl include, but are not limited to, cyclopentyl, cyclohexyl,1-cyclohexenyl, 3-cyclohexenyl, cycloheptyl, and the like. Examples ofheterocycloalkyl include, but are not limited to,1-(1,2,5,6-tetrahydropyridyl), 1-piperidinyl, 2-piperidinyl,3-piperidinyl, 4-morpholinyl, 3-morpholinyl, tetrahydrofuran-2-yl,tetrahydrofuran-3-yl, tetrahydrothien-2-yl, tetrahydrothien-3-yl,1-piperazinyl, 2-piperazinyl, and the like.

The term “aryl” means, unless otherwise stated, a polyunsaturated,aromatic moiety that can be a single ring or multiple rings (preferablyfrom 1 to 3 rings), which are fused together or linked covalently. Theterm “heteroaryl” refers to aryl groups (or rings) that contain from oneto four heteroatoms selected from N, O, S, and Se, wherein the nitrogen,sulfur, and selenium atoms are optionally oxidized, and the nitrogenatom(s) are optionally quaternized. A heteroaryl group can be attachedto the remainder of the molecule through a heteroatom. Non-limitingexamples of aryl and heteroaryl groups include phenyl, 1-naphthyl,2-naphthyl, 4-biphenyl, 1-pyrrolyl, 2-pyrrolyl, 3-pyrrolyl, 3-pyrazolyl,2-imidazolyl, 4-imidazolyl, pyrazinyl, 2-oxazolyl, 4-oxazolyl,2-phenyl-4-oxazolyl, 5-oxazolyl, 3-isoxazolyl, 4-isoxazolyl,5-isoxazolyl, 2-thiazolyl, 4-thiazolyl, 5-thiazolyl, 2-furyl, 3-furyl,2-thienyl, 3-thienyl, 2-pyridyl, 3-pyridyl, 4-pyridyl, 2-pyrimidyl,4-pyrimidyl, 5-benzothiazolyl, purinyl, 2-benzimidazolyl, 5-indolyl,1-isoquinolyl, 5-isoquinolyl, 2-quinoxalinyl, 5-quinoxalinyl,3-quinolyl, tetrazolyl, benzo[b]furanyl, benzo[b]thienyl,2,3-dihydrobenzo[1,4]dioxin-6-yl, benzo[1,3]dioxol-5-yl and 6-quinolyl.Substituents for each of the above noted aryl and heteroaryl ringsystems are selected from the group of acceptable substituents describedbelow.

For brevity, the term “aryl” when used in combination with other terms(e.g., aryloxy, arylthioxy, arylalkyl) includes both aryl and heteroarylrings as defined above. Thus, the term “arylalkyl” is meant to includethose radicals in which an aryl group is attached to an alkyl group(e.g., benzyl, phenethyl, pyridylmethyl and the like) including thosealkyl groups in which a carbon atom (e.g., a methylene group) has beenreplaced by, for example, an oxygen atom (e.g., phenoxymethyl,2-pyridyloxymethyl, 3-(1-naphthyloxy)propyl, and the like).

Each of the above terms (e.g., “alkyl,” “heteroalkyl,” “aryl” and“heteroaryl”) includes both substituted and unsubstituted forms of theindicated radical. Preferred substituents for each type of radical areprovided below.

Substituents for the alkyl and heteroalkyl radicals (including thosegroups often referred to as alkylene, alkenyl, heteroalkylene,heteroalkenyl, alkynyl, cycloalkyl, heterocycloalkyl, cycloalkenyl, andheterocycloalkenyl) are generically referred to as “alkyl groupsubstituents,” and they can be one or more of a variety of groupsselected from, but not limited to: —OR′, ═O, ═NR′, ═N—OR′, —NR′R″, —SR′,-halogen, —SiR′R″R′″, —OC(O)R′, —C(O)R′, —CO₂R′, —CONR′R″, —OC(O)NR′R″,—NR″C(O)R′, —NR′—C(O)NR″R′″, —NR″C(O)₂R′, —NR—C(NR′R″R′″)═NR″″,—NR—C(NR′R″)═NR′″, —S(O)R′, —S(O)₂R′, —S(O)₂NR′R″, —NRSO₂R′, —CN and—NO₂ in a number ranging from zero to (2m′+1), where m′ is the totalnumber of carbon atoms in such radical. R′, R″, R′″ and R″″ eachpreferably independently refer to hydrogen, substituted or unsubstitutedheteroalkyl, substituted or unsubstituted aryl, e.g., aryl substitutedwith 1-3 halogens, substituted or unsubstituted alkyl, alkoxy orthioalkoxy groups, or arylalkyl groups. When a compound includes morethan one R group, for example, each of the R groups is independentlyselected as are each R′, R″, R′″ and R″″ groups when more than one ofthese groups is present. When R′ and R″ are attached to the samenitrogen atom, they can be combined with the nitrogen atom to form a 5-,6-, or 7-membered ring. For example, —NR′R″ is meant to include, but notbe limited to, 1-pyrrolidinyl and 4-morpholinyl. From the abovediscussion of substituents, one of skill in the art will understand thatthe term “alkyl” is meant to include groups including carbon atoms boundto groups other than hydrogen groups, such as haloalkyl (e.g., —CF₃ and—CH₂CF₃) and acyl (e.g., —C(O)CH₃, —C(O)CF₃, —C(O)CH₂OCH₃, and thelike).

Similar to the substituents described for the alkyl radical,substituents for the aryl and heteroaryl groups are generically referredto as “aryl group substituents.” The substituents are selected from, forexample: halogen, —OR′, ′O, ═NR′, ═N—OR′, —NR′R″, —SR′, -halogen,—SiR′R″R′″, —OC(O)R′, —C(O)R′, —CO₂R′, —CONR′R″, —OC(O)NR′R″,—NR″C(O)R′, —NR′—C(O)NR″R′″, —NR″C(O)₂R′, —NR—C(NR′R″R′″)═NR″″,—NR—C(NR′R″)═NR′″, —S(O)R′, —S(O)₂R′, —S(O)₂NR′R″, —NRSO₂R′, —CN and—NO₂, —R′, —N₃, —CH(Ph)₂, fluoro(C₁-C₄)alkoxy, and fluoro(C₁-C₄)alkyl,in a number ranging from zero to the total number of open valences onthe aromatic ring system; and where R′, R″, R′″ and R″″ are preferablyindependently selected from hydrogen, substituted or unsubstitutedalkyl, substituted or unsubstituted heteroalkyl, substituted orunsubstituted aryl and substituted or unsubstituted heteroaryl. When acompound includes more than one R group, for example, each of the Rgroups is independently selected as are each R′, R″, R′″ and R″″ groupswhen more than one of these groups is present. In the schemes thatfollow, the symbol X represents “R” as described above.

Two of the substituents on adjacent atoms of the aryl or heteroaryl ringmay optionally be replaced with a substituent of the formula-T-C(O)—(CRR′)_(q)—U—, wherein T and U are independently —NR—, —O—,—CRR′— or a single bond, and q is an integer of from 0 to 3.Alternatively, two of the substituents on adjacent atoms of the aryl orheteroaryl ring may optionally be replaced with a substituent of theformula -A-(CH₂)_(r)—B—, wherein A and B are independently —CRR′—, —O—,—NR—, —S—, —S(O)—, —S(O)₂—, —S(O)₂NR′— or a single bond, and r is aninteger of from 1 to 4. One of the single bonds of the new ring soformed may optionally be replaced with a double bond. Alternatively, twoof the substituents on adjacent atoms of the aryl or heteroaryl ring mayoptionally be replaced with a substituent of the formula—(CRR′)_(s)—X—(CR″R′″)_(d)—, where s and d are independently integers offrom 0 to 3, and X is —O—, —NR′—, —S—, —S(O)—, —S(O)₂—, or —S(O)₂NR′—.The substituents R, R′, R″ and R′″ are preferably independently selectedfrom hydrogen or substituted or unsubstituted (C₁-C₆)alkyl.

As used herein, the term “heteroatom” includes oxygen (O), nitrogen (N),sulfur (S), phosphorus (P), silicon (Si), and selenium (Se).

The term “amino” or “amine group” refers to the group —NR′R″ (orN⁺RR′R″) where R, R′ and R″ are independently selected from the groupconsisting of hydrogen, alkyl, substituted alkyl, aryl, substitutedaryl, aryl alkyl, substituted aryl alkyl, heteroaryl, and substitutedheteroaryl. A substituted amine being an amine group wherein R′ or R″ isother than hydrogen. In a primary amino group, both R′ and R″ arehydrogen, whereas in a secondary amino group, either, but not both, R′or R″ is hydrogen. In addition, the terms “amine” and “amino” caninclude protonated and quaternized versions of nitrogen, comprising thegroup —N⁺RR′R″ and its biologically compatible anionic counterions.

The term “aqueous solution” as used herein refers to a solution that ispredominantly water and retains the solution characteristics of water.Where the aqueous solution contains solvents in addition to water, wateris typically the predominant solvent.

The term “Carboxyalkyl” as used herein refers to a group having thegeneral formula —(CH₂)_(n)COOH wherein n is 1-18.

The term “activated alkyne,” as used herein, refers to a chemical moietythat selectively reacts with an alkyne reactive group, such as an azidomoiety or an phosphine moiety, on another molecule to form a covalentchemical bond between the activated alkyne group and the alkyne reactivegroup. Examples of alkyne-reactive groups include azides.“Alkyne-reactive” can also refer to a molecule that contains a chemicalmoiety that selectively reacts with an alkyne group. As used hereinactivated alkyne encompasses any terminal alkynes or cyclooctynes(dipolarophiles) that will react with 1,3-dipoles such as azides in afacile fashion.

The term “aqueous solution,” as used herein, refers to a solution thatis predominantly water and retains the solution characteristics ofwater. Where the aqueous solution contains solvents in addition towater, water is typically the predominant solvent.

The term “azide reactive,” as used herein, refers to a chemical moietythat selectively reacts with an azido modified group on another moleculeto form a covalent chemical bond between the azido modified group andthe azide reactive group. Examples of azide-reactive groups includealkynes and phospines (e.g. triaryl phosphine). “Azide-reactive” canalso refer to a molecule that contains a chemical moiety thatselectively reacts with an azido group.

The term “buffer,” as used herein, refers to a system that acts tominimize the change in acidity or basicity of the solution againstaddition or depletion of chemical substances.

The term “carrier molecule,” as used herein, refers to a biological or anon-biological component that is covalently bonded to compound of thepresent invention. Such components include, but are not limited to, anamino acid, a peptide, a protein, a polysaccharide, a nucleoside, anucleotide, an oligonucleotide, a nucleic acid, a hapten, a psoralen, adrug, a hormone, a lipid, a lipid assembly, a synthetic polymer, apolymeric microparticle, a biological cell, a virus and combinationsthereof.

The term, “chemical handle” as used herein refers to a specificfunctional group, such as an azide, alkyne, activated alkyne, phosphite,phosphine, and the like. The chemical handle is distinct from thereactive group, defined below, in that the chemical handle are moietiesthat are rarely found in naturally-occurring biomolecules and arechemically inert towards biomolecules (e.g, native cellular components),but when reacted with an azide- or alkyne-reactive group the reactioncan take place efficiently under biologically relevant conditions (e.g.,cell culture conditions, such as in the absence of excess heat or harsereactants).

The term “click chemistry,” as used herein, refers to the Huisgencycloaddition or the 2,3-dipolar cycloaddition between an azide and aterminal alkyne to form a 1,2,4-triazole. Such chemical reactions canuse, but are not limited to, simple heteroatomic organic reactants andare reliable, selective, stereospecific, and exothermic.

The term “cycloaddition” as used herein refers to a chemical reaction inwhich two or more π-electron systems (e.g., unsaturated molecules orunsaturated parts of the same molecule) combine to form a cyclic productin which there is a net reduction of the bond multiplicity. In acycloaddition, the π electrons are used to form new sigma bonds. Theproduct of a cycloaddition is called an “adduct” or “cycloadduct”.Different types of cycloadditions are known in the art including, butnot limited to, [3+2] cycloadditions and Diels-Alder reactions. [3+2]cycloadditions, which are also called 2,3-dipolar cycloadditions, occurbetween a 1,3-dipole and a dipolarophile and are typically used for theconstruction of five-membered heterocyclic rings. The terms “[3+2]cycloaddition” also encompasses “copperless” [3+2] cycloadditionsbetween azides and cyclooctynes and difluorocyclooctynes described byBertozzi et al. J. Am. Chem. Soc., 2004, 126:15046-15047.

The term “detectable response” as used herein refers to an occurrenceof, or a change in, a signal that is directly or indirectly detectableeither by observation or by instrumentation. Typically, the detectableresponse is an occurrence of a signal wherein the fluorophore isinherently fluorescent and does not produce a change in signal uponbinding to a metal ion or biological compound. Alternatively, thedetectable response is an optical response resulting in a change in thewavelength distribution patterns or intensity of absorbance orfluorescence or a change in light scatter, fluorescence lifetime,fluorescence polarization, or a combination of the above parameters.Other detectable responses include, for example, chemiluminescence,phosphorescence, radiation from radioisotopes, magnetic attraction, andelectron density.

The term “detectably distinct” as used herein refers to a signal that isdistinguishable or separable by a physical property either byobservation or by instrumentation. For example, a fluorophore is readilydistinguishable either by spectral characteristics or by fluorescenceintensity, lifetime, polarization or photo-bleaching rate from anotherfluorophore in the sample, as well as from additional materials that areoptionally present.

The term “directly detectable” as used herein refers to the presence ofa material or the signal generated from the material is immediatelydetectable by observation, instrumentation, or film without requiringchemical modifications or additional substances.

The term “fluorophore” as used herein refers to a composition that isinherently fluorescent or demonstrates a change in fluorescence uponbinding to a biological compound or metal ion, i.e., fluorogenic.Fluorophores may contain substitutents that alter the solubility,spectral properties or physical properties of the fluorophore. Numerousfluorophores are known to those skilled in the art and include, but arenot limited to coumarin, cyanine, benzofuran, a quinoline, aquinazolinone, an indole, a benzazole, a borapolyazaindacene andxanthenes including fluoroscein, rhodamine and rhodol as well as otherfluorophores described in RICHARD P. HAUGLAND, MOLECULAR PROBES HANDBOOKOF FLUORESCENT PROBES AND RESEARCH CHEMICALS (10^(th) edition, CD-ROM,September 2005), which is herein incorporated by reference in itsentirety.

The term “isolated”, when used herein in reference to a nucleic acidpolymer, means a nucleic acid polymer, which by virtue of its origin ormanipulation is separated from at least some of the components withwhich it is naturally associated or with which it is associated wheninitially obtained. By “isolated”, it is alternatively or additionallymeant that the nucleic acid polymer of interest is produced orsynthesized by the hand of man.

The term “kit,” as used herein, refers to a packaged set of relatedcomponents, typically one or more compounds or compositions.

The term “label,” as used herein, refers to a chemical moiety or proteinthat is directly or indirectly detectable (e.g. due to its spectralproperties, conformation or activity) when attached to a target orcompound and used in the present methods, including reporter moleculesand carrier molecules. The label can be directly detectable(fluorophore) or indirectly detectable (hapten or enzyme). Such labelsinclude, but are not limited to, radiolabels that can be measured withradiation-counting devices; pigments, dyes or other chromogens that canbe visually observed or measured with a spectrophotometer; spin labelsthat can be measured with a spin label analyzer; and fluorescent labels(fluorophores), where the output signal is generated by the excitationof a suitable molecular adduct and that can be visualized by excitationwith light that is absorbed by the dye or can be measured with standardfluorometers or imaging systems, for example. The label can be achemiluminescent substance, where the output signal is generated bychemical modification of the signal compound; a metal-containingsubstance; or an enzyme, where there occurs an enzyme-dependentsecondary generation of signal, such as the formation of a coloredproduct from a colorless substrate. The term label can also refer to a“tag” or hapten that can bind selectively to a conjugated molecule suchthat the conjugated molecule, when added subsequently along with asubstrate, is used to generate a detectable signal. For example, one canuse biotin as a tag and then use an avidin or streptavidin conjugate ofhorseradish peroxidate (HRP) to bind to the tag, and then use acolorimetric substrate (e.g., tetramethylbenzidine (TMB)) or afluorogenic substrate such as Amplex Red reagent (Molecular Probes,Inc.) to detect the presence of HRP. Numerous labels are know by thoseof skill in the art and include, but are not limited to, particles,fluorophores, haptens, enzymes and their colorimetric, fluorogenic andchemiluminescent substrates and other labels that are described inRICHARD P. HAUGLAND, MOLECULAR PROBES HANDBOOK OF FLUORESCENT PROBES ANDRESEARCH PRODUCTS (10^(th) edition, CD-ROM, September 2005), supra.

The term “linker” or “L”, as used herein, refers to a single covalentbond or a series of stable covalent bonds incorporating 1-30 nonhydrogenatoms selected from the group consisting of C, N, O, S and P. Inaddition, the linker covalently attaches a carrier molecule or solidsupport to the present azido or activated alkyne modified nucleotides ornucleic acid polymers. Exemplary linking members include a moiety thatincludes —C(O)NH—, —C(O)O—, —NH—, —S—, —O—, and the like. A “cleavablelinker” is a linker that has one or more cleavable groups that may bebroken by the result of a reaction or condition. The term “cleavablegroup” refers to a moiety that allows for release of a portion, e.g., areporter molecule, carrier molecule or solid support, of a conjugatefrom the remainder of the conjugate by cleaving a bond linking thereleased moiety to the remainder of the conjugate. Such cleavage iseither chemical in nature, or enzymatically mediated. Exemplaryenzymatically cleavable groups include natural amino acids or peptidesequences that end with a natural amino acid. In addition toenzymatically cleavable groups, it is within the scope of the presentinvention to include one or more sites that are cleaved by the action ofan agent other than an enzyme. Exemplary non-enzymatic cleavage agentsinclude, but are not limited to, acids, bases, light (e.g., nitrobenzylderivatives, phenacyl groups, benzoin esters), and heat. Many cleaveablegroups are known in the art. See, for example, Jung et al., Biochem.Biophys. Acta, 761: 152-162 (1983); Joshi et al., J. Biol. Chem., 265:14518-14525 (1990); Zarling et al., J. Immunol., 124: 913-920 (1980);Bouizar et al., Eur. J. Biochem., 155: 141-147 (1986); Park et al., J.Biol. Chem., 261: 205-210 (1986); Browning et al., J. Immunol., 143:1859-1867 (1989). Moreover a broad range of cleavable, bifunctional(both homo- and hetero-bifunctional) spacer arms are commerciallyavailable. An exemplary cleavable group, an ester, is cleavable groupthat may be cleaved by a reagent, e.g. sodium hydroxide, resulting in acarboxylate-containing fragment and a hydroxyl-containing product.

The term “nucleic acid polymer” as used herein refers to adeoxyribonucleotide or ribonucleotide polymer in either single- ordouble-stranded form, including DNA and RNA, and unless otherwise statedencompasses nucleic acid-like structures with synthetic backbones, aswell as amplification products. In the context of the present invention,a nucleic acid polymer may be an isolated molecule, present in anamplification reaction or present in a hybridization reaction.

As used herein, the term “nucleotide analogue” refers to a molecule thatis structurally similar to a natural nucleotide and that can function ina similar manner as the naturally occurring nucleotide (e.g., exhibitssimilar ability to base pair with one of the naturally occurring bases).The term “nucleoside analogue”, as used herein, refers to a moleculethat is structurally similar to a natural nucleoside and that canfunction in a similar manner as the naturally occurring nucleoside(e.g., exhibits similar ability to be incorporated into DNA by DNAreplication). The term “nucleotide” refers to a monomeric unit of DNA orRNA containing a sugar moiety (pentose), a phosphate or polyphosphateand a nitrogenous heterocyclic base. The base is linked to the sugarmoiety via the glycosidic carbon (i.e., the 1′-carbon of the pentose)and that combination of base and sugar is called a “nucleoside”. Thebase characterizes the nucleotide (or nucleoside) with the four bases ofDNA being adenine (or A), guanine (G), cytosine (C) and thymine (T), andthe four bases of RNA being adenine, guanine, cytosine, and uracil (U).In certain embodiments of the present invention a nucleotide analogue(or nucleoside analogue) comprises a chemical handle and as used hereinalso referred to as “modified nucleotides” or “modified nucleic acidpolymers”.

The term “reactive group” as used herein refers to a group that iscapable of reacting with another chemical group to form a covalent bond,i.e. is covalently reactive under suitable reaction conditions, andgenerally represents a point of attachment for another substance. Asused herein, reactive groups refer to chemical moieties generally foundin biological systems and that react under normal biological conditions,these are herein distinguished from the chemical handel, defined above,the azido and activated alkyne moieties of the present invention. Asreferred to herein the reactive group is a moiety, such as carboxylicacid or succinimidyl ester, that is capable of chemically reacting witha functional group on a different compound to form a covalent linkage.Reactive groups generally include nucleophiles, electrophiles andphotoactivatable groups.

The term “reporter molecule” refers to any moiety capable of beingattached to a carrier molecule or solid support, such as a modifiednucleotide or nucleic acid polymer, and detected either directly orindirectly. Reporter molecules include, without limitation, achromophore, a fluorophore, a fluorescent protein, a phosphorescent dye,a tandem dye, a particle, a hapten, an enzyme and a radioisotope.Preferred reporter molecules include fluorophores, fluorescent proteins,haptens, and enzymes.

The term “sample” as used herein refers to any material that may containan analyte for detection or quantification or a modified nucleotide ornucleic acid polymer. The analyte may include a reactive group, e.g., agroup through which a compound of the invention can be conjugated to theanalyte. The sample may also include diluents, buffers, detergents, andcontaminating species, debris and the like that are found mixed with thetarget. Illustrative examples include urine, sera, blood plasma, totalblood, saliva, tear fluid, cerebrospinal fluid, secretory fluids fromnipples and the like. Also included are solid, gel or sol substancessuch as mucus, body tissues, cells and the like suspended or dissolvedin liquid materials such as buffers, extractants, solvents and the like.Typically, the sample is a live cell, a biological fluid that comprisesendogenous host cell proteins, nucleic acid polymers, nucleotides,oligonucleotides, peptides and buffer solutions. The sample may be in anaqueous solution, a viable cell culture or immobilized on a solid orsemi solid surface such as a polyacrylamide gel, membrane blot or on amicroarray.

The term “solid support,” as used herein, refers to a material that issubstantially insoluble in a selected solvent system, or which can bereadily separated (e.g., by precipitation) from a selected solventsystem in which it is soluble. Solid supports useful in practicing thepresent invention can include groups that are activated or capable ofactivation to allow selected one or more compounds described herein tobe bound to the solid support.

The term “Staudinger ligation” as used herein refers to a chemicalreaction developed by Saxon and Bertozzi (E. Saxon and C. Bertozzi,Science, 2000, 287: 2007-2010) that is a modification of the classicalStaudinger reaction. The classical Staudinger reaction is a chemicalreaction in which the combination of an azide with a phosphine orphosphite produces an aza-ylide intermediate, which upon hydrolysisyields a phosphine oxide and an amine. A Staudinger reaction is a mildmethod of reducing an azide to an amine; and triphenylphosphine iscommonly used as the reducing agent. In a Staudinger ligation, anelectrophilic trap (usually a methyl ester) is appropriately placed on atriarylphosphine aryl group (usually ortho to the phosphorus atom) andreacted with the azide, to yield an aza-ylide intermediate, whichrearranges in aqueous media to produce a compound with amide group and aphosphine oxide function. The Staudinger ligation is so named because itligates (attaches/covalently links) the two starting molecules together,whereas in the classical Staudinger reaction, the two products are notcovalently linked after hydrolysis.

The terms “structural integrity of the [nucleic acid] is not reduced” or“preservation of the structural integrity of the [nucleic acid]”, asused herein, means that either: 1) when analyzed by gel electrophoresisand detection (such as staining), a band or spot arising from thelabeled nucleic acid is not reduced in intensity by more than 20%, andpreferably not reduced by more than 10%, with respect to thecorresponding band or spot arising from the same amount of theelectrophoresed unlabeled nucleic acid, arising from the labeled nucleicacid analyzed; or 2) when analyzed by gel electrophoresis, a band orspot arising from the labeled nucleic acid is not observed to besignificantly less sharp than the corresponding band or spot arisingfrom the same amount of the electrophoresed unlabeled nucleic acid,where “ significantly less sharp” (synonymous with “significantly morediffuse”) means the detectable band or spot takes up at least 5% more,preferably 10% more, more preferably 20% more area on the gel than thecorresponding unlabeled nucleic acid. Other reproducible tests forstructural integrity of labeled nucleic acids include, withoutlimitation detection of released amino acids or peptides, or massspectrometry.

In general, for ease of understanding the present invention, themetabolic and enzymatic labeling of nucleic acids with azide moieties,alkyne moieties or phosphine, and the chemical labeling of such moietieswith azide reactive moieties, alkyne reactive moieties or phosphinereactive moieties will first be described in detail. This will befollowed by some embodiments in which such labeled nucleic acids can bedetected, isolated and/or analyzed. Exemplified methods are thendisclosed.

Labeling of Nucleic Acid Polymer using [3+2] Cycloaddition

The nucleic acid polymers produced according to methods describedherein, or utilized in methods described herein, are single- ordouble-stranded deoxyribonucleotide or ribonucleotide polymers. As willbe appreciated by one of ordinary skill in the art, the nucleic acidpolymers can be polynucleotides of any of a wide range of sizesincluding short oligonucleotides comprising at least about 8 nucleotidesas well as full genomic DNA molecules.

Some of the labeling methods described herein generally include a [3+2]cycloaddition between a first reactive unsaturated group on a nucleotideincorporated into a nucleic acid polymer and a second reactiveunsaturated group attached to a reporter molecule, a carrier moleculeand/or a solid support. Thus, in one aspect the modified nucleic acidpolymer comprises an azido group with reacts with an activated alkyne ona reporter molecule, a carrier molecule and/or a solid support to form acovalent bond. In another aspect the modified nucleic acid polymercomprises an activated alkynes that reacts with an azido moiety onreporter molecule, a carrier molecule and/or a solid support to form acovalent bond.

As described herein the tagging/labeling of nucleic acid polymers, alsoreferred to herein as nucleic acids, utilize the incorporation of abioorthoganol moieties into a nucleic acid polymer followed by chemicalattachment of a label. The bioorthoganol moieties can be incorporatedinto a nucleic acid using in vitro extension and/or amplificationtechniques including but not limited to, polymerase chain reaction(PCR), ligation-based thermocycling approaches, reversetranscription-PCR, real-time PCR, linear amplification techniques andisothermal DNA amplification techniques such as, by way of example only,real-time strand displacement amplification (SDA), rolling-circleamplification (RCA), multiple-displacement amplification (MDA), Q-betareplicase amplification, automated Q-beta replicase amplification assayand other RNA polymerase mediated techniques such as, for example,nucleic acid sequence based amplification or NASBA and transcriptionmediated amplification (TMA). In certain embodiments, such bioorthoganolmoieties are incorporated using telomerase based incorporation. Thesebioorthogonol moieties are non-native, non-perturbing chemical handlespossessing unique chemical functionality that can be modified throughhighly selective reactions. Examples of such moieties includes, but isnot limited to hyrazide and aminooxy derivatives, azides that can beselectively modified with phosphines (Staudinger ligation), azides thatcan be selectively modified with activated alkynes, and azides that canbe selectively modified with terminal alkynes (“click” chemistry). Thenucleic acids in which such bioorthoganol moieties can be incorporatedinto include, but are not limited to, DNA, RNA and oligonuceotides.

In certain embodiments the isothermal DNA amplification technology usingHelicase Dependent amplification is used to incorporate bioorthoganolmoieties into a nucleic acid. Examples of such Helicase Dependentamplification include:

-   -   (a) mHDA technology which amplifies DNA at a single temperature        (37° C.) by utilizing the unwinding activity of a DNA Helicase        and a DNA synthesis activity of a DNA polymerase;    -   (b) tHDA technology which amplifies DNA at a single temperature        (65° C.) by utilizing the unwinding activity of a thermo        tolerant DNA Helicase and a DNA synthesis activity of Bst DNA        polymerase (from Bacillus stearoethermophillus);    -   (c) circular HDA in which DNA amplification uses T7 Helicase and        T7DNA polymerase and is similar to rolling circle DNA        amplification. Other accessory proteins in this platform include        T7 single strand DNA binding protein. This platform can be used        for in vitro amplification of plasmid or covalent closed        circular DNA. This technology has significant use in clinical        diagnostics and molecular biology e.g., in DNA sequencing and        mutagenesis, and    -   d) rt-HDA takes advantage of the reverse transcriptase activity        of reverse transcriptase under constant temperature conditions        combined with polymerase activity of Bst polymerase.

In certain embodiments the isothermal DNA amplification technologyStrand Displacement Amplification (SDA) is used to incorporatebioorthoganol moieties into a nucleic acid. In SDA a primer contains arestriction site is annealed to template. Amplification primers are thenannealed to 5′ adjacent sequences (form a nick) and start amplificationat a fixed temperature. Newly synthesized DNA are nicked by arestriction enzyme, polymerase starts amplification again, displacingthe newly synthesized strands. 10⁹ copies of DNA can be made in onereaction.

In certain embodiments the isothermal DNA amplification technology Loopmediated Isothermal DNA amplification is used to incorporatebioorthoganol moieties into a nucleic acid. Loop-mediated IsothermalAmplification (LAMP) uses 4 primers, which recognize 6 distinct regionson the target gene and a DNA polymerase with strand displacementactivity to carry out reaction under isothermal condition. Amplificationand detection of gene can be completed in a single step, by incubatingthe mixture of samples, primers, DNA polymerase with strand displacementactivity and substrate at a constant temperature between 60-65° C. Itprovides high amplification efficiency, with DNA being amplified109-1010 times in 15-60 minutes. Because of its high specificity, thepresence of amplified product can indicate the presence of target gene.LAMP also uses Bst DNA polymerase.

In certain embodiments, rolling circle DNA amplification/Phi29 based DNAwhole genome (or partial genome) amplification is used to incorporatebioorthoganol moieties into a nucleic acid. This method uses phi 29 DNApolymerase and can amplify DNA (Linear or circular) with high fidelityand efficiency. Such amplification methods can be use for thepreparation of DNA probes from in situ hybridizations.

Nucleic acid polymers containing at least one nucleotide analogue may bealternatively be prepared by any of a variety of methods well known inthe art including synthetic and enzymatic methods (J. Sambrook et al.,“Molecular Cloning: A Laboratory Manual”, 1989, 2^(nd) Ed., Cold SpringHarbour Laboratory Press: New York, N.Y.; “PCR Protocols: A Guide toMethods and Applications”, 1990, M. A. Innis (Ed.), Academic Press: NewYork, N.Y.; P. Tijssen “Hybridization with Nucleic AcidProbes—Laboratory Techniques in Biochemistry and Molecular Biology(Parts I and II)”, 1993, Elsevier Science; “PCR Strategies”, 1995, M. A.Innis (Ed.), Academic Press: New York, N.Y.; and “Short Protocols inMolecular Biology”, 2002, F. M. Ausubel (Ed.), 5^(th) Ed., John Wiley &Sons: Secaucus, N.J.).

Nucleic acids used in the methods described herein may be prepared usingautomated, solid-phase procedure based on the phosphoramidite approach.In such a method, each nucleotide (including nucleotide analogues) isindividually added to the 5′-end of the growing polynucleotide chain,which is attached at the 3′-end to a solid support. The addednucleotides are in the form of trivalent 3′-phosphoramidites that areprotected from polymerization by a dimethoxytriyl (or DMT) group at the5′-position. After base-induced phosphoramidite coupling, mild oxidationto give a pentavalent phosphotriester intermediate, DMT removal providesa new site for polynucleotide elongation. The nucleic acid polymers arethen cleaved off the solid support, and the phosphodiester and exocyclicamino groups are deprotected with ammonium hydroxide. These synthesesmay be performed on oligo synthesizers such as those commerciallyavailable from Perkin Elmer/Applied Biosystems, Inc (Foster City,Calif.), DuPont (Wilmington, Del.) or Milligen (Bedford, Mass.).

As will be appreciated by one of ordinary skill in the art, nucleic acidpolymers of the described herein may be prepared either by apre-synthetic modification method (i.e., incorporation of nucleotidesanalogues into the nucleic acid molecule) or a post-syntheticmodification method (i.e., modification of naturally occurringnucleotides to nucleotide analogues in the nucleic acid molecule).Alternatively, nucleotide analogues can be incorporated into the DNA ofcells or living systems by DNA replication, or into RNA by reaction, asdescribed below.

Thus, in certain embodiments are provided methods for making modifiednucleic acid polymers and the polymers themselves.

In one aspect is a method for making an azido, alkyne or phosphinemodified nucleic acid polymer, wherein the method comprises:

-   -   incubating at least one azido, alkyne or phosphine modified        nucleotide in the presence of a nucleic acid amplification        enzyme to form an azido, alkyne or phosphine modified nucleic        acid polymer.        In one aspect, the nucleic acid enzyme is a DNA polymerase and        in another aspect the nucleic acid enzyme is a RNA polymerase.        We have unexpectedly found that incorporation of azido modified        nucleotides into a nucleic acid polymer increases the melting        temperature of the polymer under hybridization conditions. Thus,        any application wherein a probes could be utilized that has an        increased melting temperature is invisioned in the present        methods of using the modified nucleic acid polymers.

Isolation or purification of the nucleic acid polymers of the presentinvention, where necessary, may be carried out by any of a variety ofmethods well-known in the art. Purification of nucleic acid polymers istypically performed either by native acrylamide gel electrophoresis,agarose electrophoresis or by size exclusion or by anion-exchange HPLCas described, for example by J. D. Pearson and F. E. Regnier (J. Chrom.,1983, 255: 137-149) or by reverse phase HPLC (G. D. McFarland and P. N.Borer, Nucleic Acids Res., 1979, 7: 1067-1080).

If desired, the sequence of synthetic nucleic acid polymers can beverified using any suitable sequencing method including, but not limitedto, chemical degradation (A. M. Maxam and W. Gilbert, Methods ofEnzymology, 1980, 65: 499-560), matrix-assisted laser desorptionionization time-of-flight (MALDI-TOF) mass spectrometry (U. Pieles etal., Nucleic Acids Res., 1993, 21: 3191-3196), mass spectrometryfollowing alkaline phosphatase and exonuclease digestions (H. Wu and H.Aboleneen, Anal. Biochem., 2001, 290: 347-352), and the like.

Provided herein are methods and compositions for detection, isolationand/or analysis of labeled nucleic acids facilitated by theincorporation of nucleotides comprising azide moieties, alkyne moieties,or phosphine moieties. In particular, presented are a novel methods forA) amplification methods for incorporating a nucleotide comprising anazide moiety into a nucleic acid, B) labeling such azido modifiednucleic acids in solution followed by separation using methods known inthe art for separating nucleic acids, C) labeling immobilized modifiednucleic acids and D) novel methods for telomerase based assays usingsuch modified nucleic acids. In addition, these azide, activated alkyneor phosphine modified nucleic acids can form conjugates with reportermolecules, carrier molecules and/or solid supports using methodsprovided herein. The reporter molecules can include, but are not limitedto labels, while the solid supports can include, but are not limited to,solid support resins, microtiter plates and microarray slides. Thecarrier molecules can include, but are not limited to, affinity tags,nucleotides, oligonucleotides and polymers. In certain embodiments, thenucleic acids are modified with alkyne containing nucleotides, and inother embodiments, the nucleic acids are modified with phosphinecontaining nucleotides.

Nucleoside and Nucleotide Analogues

Nucleoside analogues (or nucleotide analogues) suitable for use in themethods described herein include any nucleoside analogue (or nucleotideanalogue), as defined herein, that contains a reactive bioorthoganolmoiety that can undergo a [3+2] cycloaddition or Staudinger ligation. Insome embodiments, the reactive bioorthoganol moiety is carried by thebase of the nucleoside (or nucleotide). The base carrying the reactivebioorthoganol moiety can be a purine (e.g., adenine or guanine) or apyrimidine (e.g., cytosine, uracil or thymine). In certain embodiments,the base is uracil; in some such embodiments, uracil carries thereactive bioorthoganol moiety on the 5-position. In certain embodiments,the base is adenine; in some such embodiments, adenine carries thereactive bioorthoganol moiety. In certain embodiments, the bioorthoganolmoiety is indirectly attached to the base, while in other embodimentsthe bioorthoganol moiety is directly covalently attached to the base.Non-limiting examples of the nucleoside analogues that may be used inthe methods described herein include 5-ethynyl-2′deoxyuracil (alsotermed herein ethynyluracil or EdU) and 5-azido-2′-deoxyuracil (alsotermed herein azidouracil or AdU) as well as their triphosphate andphosphoramidite forms. EdU can be synthesized essentially as describedby C.-S. Yu and F. Oberdorfer, Synlett, 2000, 1: 86-88; and AdU can besynthesized using a method similar to that described in P. Sunthankar etal., Anal. Biochem., 1998, 258: 195-201 to synthesize azido-dUMP. EdU isalso commercially available from Berry and Associates, Inc. (Dexter,Mich.).

In certain embodiments, the reactive bioorthoganol moiety is carried bythe sugar (ribose and deoxyribose) of the nucleoside (or nucleotide). Incertain embodiments, the bioorthoganol moiety is indirectly attached tothe sugar, while in other embodiments the bioorthoganol moiety isdirectly covalently attached to the sugar. In certain embodiments, thenucleotide is a nucleotide monophosphate with the reactive bioorthogalmoiety attached to the phosphate moiety. In certain embodiments, thenucleotide is a nucleotide diphosphate with the reactive bioorthoganolmoiety attached to the terminal phosphate moiety. In certainembodiments, the nucleotide is a nucleotide triphosphate with thereactive bioorthoganol moiety attached to the terminal phosphate moiety.The sugar carrying the reactive bioorthoganol moiety can covalentlyattached to a purine (e.g., adenine or guanine) or a pyrimidine (e.g.,cytosine, uracil or thymine). In certain embodiments, the base isuracil, while in other embodiments the base is adenine. Non-limitingexamples of the nucleotide triphosphate analogues that may be used inthe methods described herein (see FIG. 1) include N₃-dATP (azide-dATP),N₃-dUTP (azide-dUTP), N₃-dTTP, N₃-dGTP, N₃-dCTP, E-dATP (ethynyl-dATP)and E-dUTP (ethynyl-dUTP), E-dGTP, E-dCTP, E-dTTP.

The reactive bioorthoganol moiety can be a 1,3-dipole such as a nitrileoxide, an azide, a diazomethane, a nitrone or a nitrile imine. Incertain embodiments, the 1,3-dipole is an azide. Alternatively, thereactive bioorthoganol moiety can be a dipolarophile such as an alkene(e.g., vinyl, propylenyl, and the like) or an alkyne (e.g., ethynyl,propynyl, and the like). In certain embodiments, the dipolarophile is analkyne, such as, for example, an ethynyl group.

Chemical modification of Nucleic Acids Containing Azide, Alkyne orPhosphine Moieties

The nucleic acids that can be chemically modified using the methodsdescribed herein contain azide moieties, alkyne moieties or phosphinemoieties that are incorporated into nucleic acids using variousamplification techniques utilizing nucleobases that contain azidemoieties, alkyne moieties or phosphine moieties. Such nucleobases havebeen chemical synthesized as described herein. These azide moieties,alkyne moieties and phosphine moieties are non-native, non-perturbingbioorthogonol chemical moieties that possess unique chemicalfunctionality that can be modified through highly selective reactions.Non-limiting examples of such reactions used in the methods describedherein are shown in FIG. 2, wherein the chemical labeling of nucleicacids that contain azide moieties or alkyne moieties utilizeCopper(I)-catalyzed Azide-Alkyne Cycloaddition, also referred to hereinas “click” chemistry, the chemical labeling of nucleic acids thatcontain azide moieties or phosphine moieties utilize Staudingerligation, and the chemical labeling of nucleic acids that containactivated-alkyne moieties or activated-alkyne reactive moieties.

“Click” Chemistry

Azides and terminal or internal alkynes can undergo a 1,3-dipolarcycloaddition (Huisgen cycloaddition) reaction to give a 1,2,3-triazole.However, this reaction requires long reaction times and elevatedtemperatures. Alternatively, azides and terminal alkynes can undergoCopper(I)-catalyzed Azide-Alkyne Cycloaddition (CuAAC) at roomtemperature. Such copper(I)-catalyzed azide-alkyne cycloadditions, alsoknown as “click” chemistry, is a variant of the Huisgen 1,3-dipolarcycloaddition wherein organic azides and terminal alkynes react to give1,4-regioisomers of 1,2,3-triazoles. Examples of “click” chemistryreactions are described by Sharpless et al. (U.S. Patent ApplicationPublication No. 20050222427, published Oct. 6, 2005, PCT/US03/17311;Lewis W G, et al., Angewandte Chemie-Int'l Ed. 41 (6): 1053; methodreviewed in Kolb, H. C., et al., Angew. Chem. Inst. Ed. 2001,40:2004-2021), which developed reagents that react with each other inhigh yield and with few side reactions in a heteroatom linkage (asopposed to carbon-carbon bonds) in order to create libraries of chemicalcompounds. As described herein, “click” chemistry is used in the methodsfor labeling nucleic acids.

The copper used as a catalyst for the “click chemistry reaction used inthe methods described herein to conjugate a label (reporter group, solidsupport or carrier molecule) to a nucleic acid is in the Cu (I)reduction state. The sources of copper(I) used in suchcopper(I)-catalyzed azide-alkyne cycloadditions can be any cuprous saltincluding, but not limited to, cuprous halides such as cuprous bromideor cuprous iodide. However, this regioselective cycloaddition can alsobe conducted in the presence of a metal catalyst and a reducing agent.In certain embodiments, copper can be provided in the Cu (II) reductionstate (for example, as a salt, such as but not limited to Cu(NO₃)₂Cu(OAc)₂ or CuSO₄), in the presence of a reducing agent wherein Cu(I) isformed in situ by the reduction of Cu(II). Such reducing agents include,but are not limited to, ascorbate, Tris(2-Carboxyethyl)Phosphine (TCEP),2,4,6-trichlorophenol (TCP), NADH, NADPH, thiosulfate, metallic copper,quinone, hydroquinone, vitamin K₁, glutathione, cysteine,2-mercaptoethanol, dithiothreitol, Fe¹⁺, Co²⁺, or an applied electricpotential. In other embodiments, the reducing agents include metalsselected from Al, Be, Co, Cr, Fe, Mg, Mn, Ni, Zn, Au, Ag, Hg, Cd, Zr,Ru, Fe, Co, Pt, Pd, Ni, Rh, and W.

The copper(I)-catalyzed azide-alkyne cycloadditions for labeling nucleicacids can be performed in water and a variety of solvents, includingmixtures of water and a variety of (partially) miscible organic solventsincluding alcohols, dimethyl sulfoxide (DMSO), dimethyl formamide (DMF),tert-butanol (tBuOH) and acetone.

Without limitation to any particular mechanism, copper in the Cu (I)state is a preferred catalyst for the copper(I)-catalyzed azide-alkynecycloadditions, or “click” chemistry reactions, used in the methodsdescribed herein. Certain metal ions are unstable in aqueous solvents,by way of example Cu(I), therefore stabilizing ligands/chelators can beused to improve the reaction. In certain embodiments at least one copperchelator is used in the methods described herein, wherein such chelatorsbinds copper in the Cu (I) state. In certain embodiments at least onecopper chelator is used in the methods described herein, wherein suchchelators binds copper in the Cu(II) state. In certain embodiments, thecopper (I) chelator is a 1,10 phenanthroline-containing copper (I)chelator. Non-limiting examples of such phenanthroline-containing copper(I) chelators include, but are not limited to, bathophenanthrolinedisulfonic acid (4,7-diphenyl-1,10-phenanthroline disulfonic acid) andbathocuproine disulfonic acid (BCS;2,9-dimethyl-4,7-diphenyl-1,10-phenanthroline disulfonate). Otherchelators used in such methods include, but are not limited to,N-(2-acetamido)iminodiacetic acid (ADA), pyridine-2,6-dicarboxylic acid(PDA), S-carboxymethyl-L-cysteine (SCMC), trientine,tetra-ehylenepolyamine (TEPA),NNNN-tetrakis(2-pyridylmethyl)ethylenediamine (TPEN), EDTA, neocuproine,N-(2-acetamido)iminodiacetic acid (ADA), pyridine-2,6-dicarboxylic acid(PDA), S-carboxymethyl-L-cysteine (SCMC),tris-(benzyl-triazolylmethyl)amine (TBTA), or a derivative thereof. Mostmetal chelators, a wide variety of which are known in the chemical,biochemical, and medical arts, are known to chelate several metals, andthus metal chelators in general can be tested for their function in 1,3cycloaddition reactions catalyzed by copper. In certain embodiments,histidine is used as a chelator, while in other embodiments glutathioneis used as a chelator and a reducing agent.

The concentration of the reducing agents used in the “click” chemistryreaction described herein can be in the micromolar to millimolar range.In certain embodiments the concentration of the reducing agent is fromabout 100 micromolar to about 100 millimolar. In other embodiments theconcentration of the reducing agent is from about 10 micromolar to about10 millimolar. In other embodiments the concentration of the reducingagent is from about 1 micromolar to about 1 millimolar.

In certain embodiments, the methods describe herein for labeling nucleicacids using “click” chemistry, at least one copper chelator is addedafter copper(II) used in the reaction has been contacted with a reducingagent. In other embodiments, at least one copper chelator can be addedimmediately after contacting copper(II) with a reducing agent. In otherembodiments, the copper chelator(s) is added between about five secondsand about twenty-four hours after copper(II) and a reducing agent havebeen combined in a reaction mixture. In other embodiments, at least onecopper chelator can be added any time to a reaction mixture thatincludes copper(II) and a reducing agent, such as, by way of exampleonly, immediately after contacting copper(II) and a reducing agent, orwithin about five minutes of contacting copper(II) and a reducing agentin the reaction mixture. In some embodiments, at least one copperchelator can be added between about five seconds and about one hour,between about one minute and about thirty minutes, between about fiveminutes and about one hour, between about thirty minutes and about twohours, between about one hour and about twenty-four hours, between aboutone hour and about five hours, between about two hours and about eighthours, after copper(II) and a reducing agent have been combined for usein a reaction mixture.

In other embodiments, one or more copper chelators can be added morethan once to such “click” chemistry reactions. In embodiments in whichmore than one copper chelator is added to a reaction, two or more of thecopper chelators can bind copper in the Cu (I) state or, one or more ofthe copper chelators can bind copper in the Cu (I) state and one or moreadditional chelators can bind copper in the Cu (II) state. In certainembodiments, one or more copper chelators can be added after the initialaddition of a copper chelator to the “click” chemistry reaction. Incertain embodiments, the one or more copper chelators added after theinitial addition of a copper chelator to the reaction can be the same ordifferent from a copper chelator added at an earlier time to thereaction.

The concentration of a copper chelator used in the “click” chemistryreaction described herein can be determined and optimized using methodswell known in the art, including those disclosed herein using “click”chemistry to label nucleic acids followed by detecting such labelednucleic acids to determine the efficiency of the labeling reaction andthe integrity of the labeled nucleic acid(s). In certain embodiments,the chelator concentrations used in the methods described herein is inthe micromolar to millimolar range, by way of example only, from 1micromolar to 100 millimolar. In certain embodiments the chelatorconcentration is from about 10 micromolar to about 10 millimolar. Inother embodiments the chelator concentration is from about 50 micromolarto about 10 millimolar. In other embodiments the chelator, can beprovided in a solution that includes a water miscible solvent such as,alcohols, dimethyl sulfoxide (DMSO), dimethyl formamide (DMF),tert-butanol (tBuOH) and acetone. In other embodiments the chelator, canbe provided in a solution that includes a solvent such as, for example,dimethyl sulfoxide (DMSO) or dimethylformamide (DMF).

In certain embodiments of the methods for labeling nucleic acidsutilizing “click” chemistry described herein, the nucleic acid canpossess an azide moiety, whereupon the label possesses an alkyne moiety,whereas in other embodiments the nucleic acid can possess an alkynemoiety, and the label possesses an azide moiety.

In certain embodiments are provided methods for forming a modifiednucleic acid polymer conjugate, wherein the method comprises:

incorporating an azide modified nucleotide into the nucleic acid polymerby contacting the azide modified nucleotide nucleotide with at least oneother nucleotide in the presence of a DNA amplification enzyme to forman azide modified nucleic acid polymer; and

contacting the azide modified nucleic acid polymer with a reportermolecule, carrier molecule or solid support that comprises an activatedor terminal alkyne or phosphine moiety to form a nucleic acidpolymer-reporter molecule, carrier molecule, solid support conjugate.

In an alternative embodiment are provided methods for forming a modifiednucleic acid polymer conjugate, wherein the method comprises:

incorporating a terminal alkyne modified nucleotide into the nucleicacid polymer by contacting the terminal alkyne modified nucleotide withat least one other nucleotide in the presence of a DNA amplificationenzyme to form a terminal alkyne modified nucleic acid polymer; and

contacting the terminal alkyne modified nucleic acid polymer with areporter molecule, carrier molecule or solid support that comprises anazido moiety to form a nucleic acid polymer-reporter molecule, carriermolecule, solid support conjugate.

Staudinger Ligation

The Staudinger reaction, which involves reaction between trivalentphosphorous compounds and organic azides (Staudinger et al. Helv. Chim.Acta 1919, 2, 635), has been used for a multitude of applications.(Gololobov et al. Tetrahedron 1980, 37, 437); (Gololobov et al.Tetrahedron 1992, 48, 1353). There are almost no restrictions on thenature of the two reactants. The Staudinger ligation is a modificationof the Staudinger reaction in which an electrophilic trap (usually amethyl ester) is placed on a triaryl phosphine. In the Staudingerligation, the aza-ylide intermediate rearranges, in aqueous media, toproduce an amide linkage and the phosphine oxide, ligating the twomolecules together, whereas in the Staudinger reaction the two productsare not covalently linked after hydrolysis. Such ligations have beendescribed in U.S. Patent Application No. 20060276658. In certainembodiments, the phosphine can have a neighboring acyl group such as anester, thioester or N-acyl imidazole (i.e. a phosphinoester,phosphinothioester, phosphinoimidazole) to trap the aza-ylideintermediate and form a stable amide bond upon hydrolysis. In certainembodiments, the phosphine can be a di- or triarylphosphine to stabilizethe phosphine. The phosphines used in the Staudinger ligation methodsdescribed herein to conjugate a label to a nucleic acid include, but arenot limited to, cyclic or acyclic, halogenated, bisphosphorus, or evenpolymeric. Similarly, the azides can be alkyl, aryl, acyl or phosphoryl.In certain embodiments, such ligations are carried out under oxygen-freeanhydrous conditions.

In certain embodiments of the methods for labeling nucleic acidutilizing Staudinger ligation described herein, the nucleic acid canpossess an azide moiety, whereupon the label possesses a phosphinemoiety, whereas in other embodiments the nucleic acid can possess aphosphine moiety, and the label possesses an azide moiety.

In certain embodiments are provided methods for forming a modifiednucleic acid polymer conjugate, wherein the methods comprises:

incorporating a phosphine modified nucleotide into the nucleic acidpolymer by contacting the phosphine modified nucleotide nucleotide withat least one other nucleotide in the presence of a DNA amplificationenzyme to form a phosphine modified nucleic acid polymer; and

contacting the phosphine modified nucleic acid polymer with a reportermolecule, carrier molecule or solid support that comprises an azidomoiety to form a nucleic acid polymer-reporter molecule, carriermolecule, solid support conjugate.

Activated-Alkyne Chemistry

Azides and alkynes can undergo catalyst-free [3+2] cycloaddition by ausing the reaction of activated alkynes with azides. Such catalyst free[3+2] cycloaddition can be used in methods described herein to conjugatea label (reporter molecule, solid support or carrier molecule) to anucleic acid. Alkynes can be activated by ring strain such as, by way ofexample only, eight membered ring structures, appendingelectron-withdrawing groups to such alkyne rings, or alkynes can beactivated by the addition of a Lewis acid such as, by way of exampleonly, Au(I) or Au(III).

In certain embodiments of the methods for labeling nucleic acidsutilizing activated alkynes described herein, the nucleic acid canpossess an azide moiety, whereupon the label possesses an activatedalkyne moiety, whereas in other embodiments the nucleic acid can possessan activated alkyne moiety, and the label possesses an azide moiety.

After nucleic acids have been modified with azide moieties, alkynemoieties or phosphine moieties, they can be reacted under appropriateconditions to form conjugates with reporter molecules, solid supports orcarrier molecules. In the methods and compositions described herein theazide moiety, alkyne moiety or phosphine moiety is used as a reactivefunctional group or chemical handle on the modified nucleic acid whereinan azide reactive moiety on a reporter molecule, a solid support or acarrier molecule, or an alkyne reactive moiety on a reporter molecule, asolid support or a carrier molecule, or a phosphine reactive moiety on areporter molecule, a solid support or a carrier molecule is reacted withthe modified nucleic acid to form a covalent conjugate comprising thenucleic acid and at least one reporter molecule, at least one solidsupport and/or at least one carrier molecule.

In certain embodiments, two azide-reactive groups are used to labelnucleic acids: the first is an alkyne moiety used in a “click” chemistryreaction, and the second is a phosphine, such as a triarylphosphine,used in a Staudinger ligation. In one embodiment, “click” chemistry isutilized to form a conjugate with a nucleic acid polymer containing anazide moiety and a reporter molecule, solid support or carrier molecule,wherein the reporter molecule, solid support and carrier moleculecontain an alkyne moiety. In another embodiment, “click” chemistry isutilized to form a conjugate with a nucleic acid polymer containing analkyne moiety and a reporter molecule, solid support and/or carriermolecule, wherein the reporter molecule, solid support and carriermolecule contain an azide moiety. In another embodiment, a Staudingerligation is utilized to form a conjugate with a nucleic acid polymercontaining an azide moiety and a reporter molecule, solid support and/orcarrier molecule, wherein the reporter molecule, solid support andcarrier molecule contain an triaryl phosphine moiety. In anotherembodiment, a Staudinger ligation is utilized to form a conjugate with anucleic acid polymer containing a triaryl phosphine moiety and areporter molecule, solid support and/or carrier molecule, wherein thereporter molecule, solid support and carrier molecule contain an azidemoiety. The methods described herein are not intended to be limited tothese two azide-reactive groups, or chemical reactions, but it isenvisioned that any chemical reaction utilizing an azide-reactive groupattached to a reporter molecule, solid support or carrier molecule canbe used with the azide, alkyne or phosphine modified nucleic acidpolymers described herein.

In certain embodiments are provided methods for forming a modifiednucleic acid polymer conjugate, wherein the method comprises:

incorporating an azide modified nucleotide into the nucleic acid polymerby contacting the azide modified nucleotide nucleotide with at least oneother nucleotide in the presence of a DNA amplification enzyme to forman azide modified nucleic acid polymer; and

contacting the azide modified nucleic acid polymer with a reportermolecule, carrier molecule or solid support that comprises an activatedor terminal alkyne or phosphine moiety to form a nucleic acidpolymer-reporter molecule, carrier molecule, solid support conjugate.

In an alternative embodiment are provided methods for forming a modifiednucleic acid polymer conjugate, wherein the method comprises:

incorporating a terminal alkyne modified nucleotide into the nucleicacid polymer by contacting the terminal alkyne modified nucleotide withat least one other nucleotide in the presence of a DNA amplificationenzyme to form a terminal alkyne modified nucleic acid polymer; and

contacting the terminal alkyne modified nucleic acid polymer with areporter molecule, carrier molecule or solid support that comprises anazido moiety to form a nucleic acid polymer-reporter molecule, carriermolecule, solid support conjugate.

The methods, as described herein, that utilize cycloaddition reactionsto label nucleic acids can be carried out at room temperature in aqueousconditions with excellent regioselectivity by the addition of catalyticamounts of Cu(I) salts to the reaction mixture. See, e.g., Tomoe, etal., (2002) Org. Chem. 67:3057-3064; and, Rostovtsev, et al., (2002)Angew. Chem. Int. Ed. 41:2596-2599. The resulting five-membered ringresulting from “click” chemistry cycloaddition is not generallyreversible in reducing environments and is stable against hydrolysis forextended periods in aqueous environments. Thus, nucleic acids attachedto a labeling agent, a detection agent, a reporter molecule, a solidsupport or a carrier molecule via such five-membered ring are stable.

The reporter molecules, solid supports and carrier molecules used in themethods and compositions described herein, can contain at least onealkyne moiety or at least one phosphine moiety capable of reacting withan azide moiety. The reporter molecules, solid supports and carriermolecules used in the methods and compositions described herein, cancontain at least one azide moiety capable of reacting with an alkynemoiety or a phosphine moiety. The reporter molecules, solid supports andcarrier molecules used in the methods and compositions described herein,can contain at least one phosphine moiety capable of reacting with anazide moiety. In certain embodiments, the phosphine moieties of thereporter molecules solid supports and carrier molecules described hereinare triarylphosphine moieties.

In certain embodiments, the reporter molecules used in the methods andcompositions described herein can include, but are not limited tolabels, while the solid supports can include, but are not limited to,solid support resins, microtiter plates and microarray slides. Thecarrier molecules can include, but are not limited to, affinity tags,nucleotides, oligonucleotides and polymers.

Reporter Molecules

The reporter molecules used in the methods and compositions providedherein include any directly or indirectly detectable reporter moleculeknown by one skilled in the art that can be covalently attached to amodified nucleic acid described herein. In certain embodiments, thereporter molecules used in the methods and compositions provided hereininclude any directly or indirectly detectable reporter molecule known byone skilled in the art that can be covalently attached to an azidemodified nucleic acid, an alkyne modified nucleic acid or a phosphinemodified nucleic acid.

Reporter molecules used in the methods and compositions described hereincan contain, but are not limited to, a chromophore, a fluorophore, afluorescent protein, a phosphorescent dye, a tandem dye, a particle, ahapten, an enzyme and a radioisotope. In certain embodiments, suchreporter molecules include fluorophores, fluorescent proteins, haptens,and enzymes.

A fluorophore used in reporter molecule in the methods and compositionsdescribed herein, can contain one or more aromatic or heteroaromaticrings, that are optionally substituted one or more times by a variety ofsubstituents, including without limitation, halogen, nitro, cyano,alkyl, perfluoroalkyl, alkoxy, alkenyl, alkynyl, cycloalkyl, arylalkyl,acyl, aryl or heteroaryl ring system, benzo, or other substituentstypically present on fluorophores known in the art.

A fluorophore used in reporter molecule in the methods and compositionsdescribed herein, is any chemical moiety that exhibits an absorptionmaximum at wavelengths greater than 280 nm, and retains its spectralproperties when covalently attached to a modified nucleotide such as, byway of example only, an azide, and alkyne or a phosphine. Fluorophoresused as in reporter molecule in the methods and compositions describedherein include, without limitation; a pyrene (including any of thecorresponding derivative compounds disclosed in U.S. Pat. No.5,132,432), an anthracene, a naphthalene, an acridine, a stilbene, anindole or benzindole, an oxazole or benzoxazole, a thiazole orbenzothiazole, a 4-amino-7-nitrobenz-2-oxa-1,3-diazole (NBD), a cyanine(including any corresponding compounds in U.S. Ser. Nos. 09/968,401 and09/969,853), a carbocyanine (including any corresponding compounds inU.S. Ser. Nos. 09/557,275; 09/969,853 and 09/968,401; U.S. Pat. Nos.4,981,977; 5,268,486; 5,569,587; 5,569,766; 5,486,616; 5,627,027;5,808,044; 5,877,310; 6,002,003; 6,004,536; 6,008,373; 6,043,025;6,127,134; 6,130,094; 6,133,445; and publications WO 02/26891, WO97/40104, WO 99/51702, WO 01/21624; EP 1 065 250 A1), a carbostyryl, aporphyrin, a salicylate, an anthranilate, an azulene, a perylene, apyridine, a quinoline, a borapolyazaindacene (including anycorresponding compounds disclosed in U.S. Pat. Nos. 4,774,339;5,187,288; 5,248,782; 5,274,113; and 5,433,896), a xanthene (includingany corresponding compounds disclosed in U.S. Pat. No. 6,162,931;6,130,101; 6,229,055; 6,339,392; 5,451,343 and U.S. Ser. No.09/922,333), an oxazine (including any corresponding compounds disclosedin U.S. Pat. No. 4,714,763) or a benzoxazine, a carbazine (including anycorresponding compounds disclosed in U.S. Pat. No. 4,810,636), aphenalenone, a coumarin (including an corresponding compounds disclosedin U.S. Pat. Nos. 5,696,157; 5,459,276; 5,501,980 and 5,830,912), abenzofuran (including an corresponding compounds disclosed in U.S. Pat.Nos. 4,603,209 and 4,849,362) and benzphenalenone (including anycorresponding compounds disclosed in U.S. Pat. No. 4,812,409) andderivatives thereof. As used herein, oxazines include resorufins(including any corresponding compounds disclosed in U.S. Pat. No.5,242,805), aminooxazinones, diaminooxazines, and theirbenzo-substituted analogs.

Xanthene type fluorophores used in reporter molecule in the methods andcompositions described herein include, but are not limited to, afluorescein, a rhodol (including any corresponding compounds disclosedin U.S. Pat. Nos. 5,227,487 and 5,442,045), or a rhodamine (includingany corresponding compounds in U.S. Pat. Nos. 5,798,276; 5,846,737; U.S.Ser. No. 09/129,015). As used herein, fluorescein includes benzo- ordibenzofluoresceins, seminaphthofluoresceins, or naphthofluoresceins.Similarly, as used herein rhodol includes seminaphthorhodafluors(including any corresponding compounds disclosed in U.S. Pat. No.4,945,171). In certain embodiments, the fluorophore is a xanthene thatis bound via a linkage that is a single covalent bond at the 9-positionof the xanthene. In other embodiments, the xanthenes include derivativesof 3H-xanthen-6-ol-3-one attached at the 9-position, derivatives of6-amino-3H-xanthen-3-one attached at the 9-position, or derivatives of6-amino-3H-xanthen-3-imine attached at the 9-position.

In certain embodiments, the fluorophores used in reporter molecules inthe methods and compositions described herein include xanthene (rhodol,rhodamine, fluorescein and derivatives thereof) coumarin, cyanine,pyrene, oxazine and borapolyazaindacene. In other embodiments, suchfluorophores are sulfonated xanthenes, fluorinated xanthenes, sulfonatedcoumarins, fluorinated coumarins and sulfonated cyanines

Non-limiting examples of the fluorophores used in reporter molecules inthe methods and compositions described herein are shown in FIG. 3,wherein such fluorphores have been modified with azide moieties, alkynemoieties or phosphine moieties. In certain embodiments, the suchfluorphores used in “click” chemistry reactions form triazole productswherein the conjugate does not requires UV excitation and any quenchingeffect due to conjugation of azido or alkyne groups to the fluorescentπ-system is overcome.

The choice of the fluorophore attached to the modified nucleic acid willdetermine the absorption and fluorescence emission properties of themodified nucleic acid. Physical properties of a fluorophore label thatcan be used for detection of modified nucleic acids include, but are notlimited to, spectral characteristics (absorption, emission and stokesshift), fluorescence intensity, lifetime, polarization andphoto-bleaching rate, or combination thereof. All of these physicalproperties can be used to distinguish one fluorophore from another, andthereby allow for multiplexed analysis. In certain embodiments, thefluorophore has an absorption maximum at wavelengths greater than 480nm. In other embodiments, the fluorophore absorbs at or near 488 nm to514 nm (particularly suitable for excitation by the output of theargon-ion laser excitation source) or near 546 nm (particularly suitablefor excitation by a mercury arc lamp). In other embodiment a fluorophorecan emit in the NIR (near infra red region) for tissue or whole organismapplications.

Many of fluorophores can also function as chromophores and thus thedescribed fluorophores are also chromophores used in reporter moleculesin the methods and compositions described herein.

In addition to fluorophores, enzymes also find use as labels for thedetection reagents/reporter molecules used in the methods andcompositions described herein. Enzymes are desirable labels becauseamplification of the detectable signal can be obtained resulting inincreased assay sensitivity. The enzyme itself does not produce adetectable response but functions to break down a substrate when it iscontacted by an appropriate substrate such that the converted substrateproduces a fluorescent, colorimetric or luminescent signal. Enzymesamplify the detectable signal because one enzyme on a labeling reagentcan result in multiple substrates being converted to a detectablesignal. This is advantageous where there is a low quantity of targetpresent in the sample or a fluorophore does not exist that will givecomparable or stronger signal than the enzyme. However, fluorophores aremost preferred because they do not require additional assay steps andthus reduce the overall time required to complete an assay. The enzymesubstrate is selected to yield the preferred measurable product, e.g.colorimetric, fluorescent or chemiluminescence. Such substrates areextensively used in the art, many of which are described in theMOLECULAR PROBES HANDBOOK, supra.

In certain embodiments, colorimetric or fluorogenic substrate and enzymecombination use oxidoreductases such as, by way of example only,horseradish peroxidase and a substrate such as, by way of example only,3,3′-diaminobenzidine (DAB) or 3-amino-9-ethylcarbazole (AEC), whichyield a distinguishing color (brown and red, respectively). Othercolorimetric oxidoreductase substrates used with the enzymatic reportermolecules described herein include, but are not limited to:2,2-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS),o-phenylenediamine (OPD), 3,3′,5,5′-tetramethylbenzidine (TMB),o-dianisidine, 5-aminosalicylic acid, 4-chloro-1-naphthol. Fluorogenicsubstrates used with the enzymatic reporter molecules described hereininclude, but are not limited to, homovanillic acid or4-hydroxy-3-methoxyphenylacetic acid, reduced phenoxazines and reducedbenzothiazines, including Amplex® Red reagent and its variants (U.S.Pat. No. 4,384,042), Amplex UltraRed and its variants in (WO05042504)and reduced dihydroxanthenes, including dihydrofluoresceins (U.S. Pat.No. 6,162,931) and dihydrorhodamines including dihydrorhodamine 123.Peroxidase substrates can be used with the enzymatic reporter moleculesdescribed herein. Such peroxide substrates include, but are not limitedto, tyramides (U.S. Pat. Nos. 5,196,306; 5,583,001 and 5,731,158) whichrepresent a unique class of peroxidase substrates in that they can beintrinsically detectable before action of the enzyme but are “fixed inplace” by the action of a peroxidase in the process described astyramide signal amplification (TSA). These substrates are extensivelyutilized to label targets in samples that are cells, tissues or arraysfor their subsequent detection by microscopy, flow cytometry, opticalscanning and fluorometry.

In other embodiments the colorimetric (and in some cases fluorogenic)substrates and enzymes combination used in reporter molecules describedherein include a phosphatase enzyme such as, by way of example only, anacid phosphatase, an alkaline phosphatase or a recombinant version ofsuch a phosphatase. A colorimetric substrate used in combination withsuch phosphatases include, but are not limited to,5-bromo-6-chloro-3-indolyl phosphate (BCIP), 6-chloro-3-indolylphosphate, 5-bromo-6-chloro-3-indolyl phosphate, p-nitrophenylphosphate, or o-nitrophenyl phosphate or with a fluorogenic substratesuch as 4-methylumbelliferyl phosphate,6,8-difluoro-7-hydroxy-4-methylcoumarinyl phosphate (DiFMUP, U.S. Pat.No. 5,830,912), fluorescein diphosphate, 3-O-methylfluoresceinphosphate, resorufin phosphate,9H-(1,3-dichloro-9,9-dimethylacridin-2-one-7-yl) phosphate (DDAOphosphate), or ELF 97, ELF 39 or related phosphates (U.S. Pat. Nos.5,316,906 and 5,443,986).

Other enzymes used in reporter molecules described herein includeglycosidases, including, but not limited to, beta-galactosidase,beta-glucuronidase and beta-glucosidase. The colorimetric substratesused with such enzymes include, but are not limited to,5-bromo-4-chloro-3-indolylbeta-D-galactopyranoside (X-gal) and similarindolyl galactosides, glucosides, and glucuronides, o-nitrophenylbeta-D-galactopyranoside (ONPG) and p-nitrophenylbeta-D-galactopyranoside. Preferred fluorogenic substrates includeresorufin beta-D-galactopyranoside, fluorescein digalactoside (FDG),fluorescein diglucuronide and their structural variants (U.S. Pat. Nos.5,208,148; 5,242,805; 5,362,628; 5,576,424 and 5,773,236),4-methylumbelliferyl beta-D-galactopyranoside, carboxyumbelliferylbeta-D-galactopyranoside and fluorinated coumarinbeta-D-galactopyranosides (U.S. Pat. No. 5,830,912).

Additional enzymes used in reporter molecules described herein includeinclude, but are not limited to, hydrolases such as cholinesterases andpeptidases, oxidases such as glucose oxidase and cytochrome oxidases,and reductases for which suitable substrates are known.

Enzymes and their appropriate substrates that produce chemiluminescencecan also be used in reporter molecules described herein. Such enzymesinclude, but are not limited to, natural and recombinant forms ofluciferases and aequorins. In addition, the chemiluminescence-producingsubstrates for phosphatases, glycosidases and oxidases such as thosecontaining stable dioxetanes, luminol, isoluminol and acridinium estersan also be used in reporter molecules described herein.

In addition to enzymes, haptens can be used in label/reporter moleculesdescribed herein. In certain embodiments, such haptens include hormones,naturally occurring and synthetic drugs, pollutants, allergens, affectormolecules, growth factors, chemokines, cytokines, lymphokines, aminoacids, peptides, chemical intermediates, nucleotides, digoxin, biotinand the like. Biotin is useful because it can function in an enzymesystem to further amplify the detectable signal, and it can function asa tag to be used in affinity chromatography for isolation purposes. Fordetection purposes, an enzyme conjugate that has affinity for biotin isused, such as, by way of example only, avidin-Horse Radish Peroxidase(HRP). Subsequently a peroxidase substrate as described herein can beadded to produce a detectable signal.

Fluorescent proteins can also be used in label/reporter moleculesdescribed herein for use in the methods, compositions and modifiednucleic acids described herein. Non-limiting examples of suchfluorescent proteins include green fluorescent protein (GFP) and thephycobiliproteins and the derivatives thereof. The fluorescent proteins,especially phycobiliprotein, are particularly useful for creating tandemdye labeled modified nucleic acids. These tandem dyes comprise afluorescent protein and a fluorophore for the purposes of obtaining alarger stokes shift wherein the emission spectra is farther shifted fromthe wavelength of the fluorescent protein's absorption spectra. This isparticularly advantageous for detecting a low quantity of a target in asample wherein the emitted fluorescent light is maximally optimized, inother words little to none of the emitted light is reabsorbed by thefluorescent protein. The fluorescent protein and fluorophore function asan energy transfer pair wherein the fluorescent protein emits at thewavelength that the fluorophore absorbs and the fluorophore then emitsat a wavelength farther from the fluorescent proteins emissionwavelength than could have been obtained with only the fluorescentprotein. A particularly useful combination is the phycobiliproteinsdisclosed in U.S. Pat. Nos. 4,520,110; 4,859,582; 5,055,556 and thesulforhodamine fluorophores disclosed in U.S. Pat. No. 5,798,276, or thesulfonated cyanine fluorophores disclosed in U.S. Ser. Nos. 09/968/401and 09/969/853; or the sulfonated xanthene derivatives disclosed in U.S.Pat. No. 6,130,101 and those combinations disclosed in U.S. Pat. No.4,542,104. Alternatively, the fluorophore functions as the energy donorand the fluorescent protein is the energy acceptor.

Carrier Molecules: Azide Reactive, Alkyne reactive and PhosphineReactive

In the methods and compositions described herein the modified nucleicacids can be conjugated to a carrier molecule. In certain embodiments,the modified nucleic acids contain at least one alkyne moiety or atleast one phosphine moiety capable of reacting with a carrier moleculecontaining an azide moiety. In other embodiments, the modified nucleicacids contain at least one azide moiety capable of reacting with acarrier molecule containing an alkyne moiety or a phosphine moiety. Inother embodiments, the modified nucleic acids contain at least onephosphine moiety capable of reacting with a carrier molecule containingan azide moiety. In certain embodiments, the phosphine moieties of themodified nucleic acids and carrier molecules are triaryl phosphinemoieties.

A variety of carrier molecules can be used in the methods andcompositions described herein, including, but not limited to, antigens,steroids, vitamins, drugs, haptens, metabolites, toxins, environmentalpollutants, amino acids, peptides, proteins, nucleic acids, nucleic acidpolymers, carbohydrates, lipids, and polymers. In certain embodiments,the carrier molecule contain an amino acid, a peptide, a protein, apolysaccharide, a nucleoside, a nucleotide, an oligonucleotide, anucleic acid, a hapten, a psoralen, a drug, a hormone, a lipid, a lipidassembly, a synthetic polymer, a polymeric microparticle, a biologicalcell, a virus or combinations thereof.

In other embodiments, the carrier molecule is selected from a hapten, anucleotide, an oligonucleotide, a nucleic acid polymer, a protein, apeptide or a polysaccharide. In still other embodiments, the carriermolecule is an amino acid, a peptide, a protein, a polysaccharide, anucleoside, a nucleotide, an oligonucleotide, a nucleic acid, a hapten,a psoralen, a drug, a hormone, a lipid, a lipid assembly, a tyramine, asynthetic polymer, a polymeric microparticle, a biological cell,cellular components, an ion chelating moiety, an enzymatic substrate ora virus. In further embodiments, the carrier molecule is an antibody orfragment thereof, an antigen, an avidin or streptavidin, a biotin, adextran, an IgG binding protein, a fluorescent protein, agarose, and anon-biological microparticle.

In certain embodiments wherein the carrier molecule is an enzymaticsubstrate, the enzymatic substrate is selected from an amino acid, apeptide, a sugar, an alcohol, alkanoic acid, 4-guanidinobenzoic acid, anucleic acid, a lipid, sulfate, phosphate, —CH₂OCO-alkyl andcombinations thereof. In certain embodiments, such enzyme substrates canbe cleaved by enzymes selected from peptidases, phosphatases,glycosidases, dealkylases, esterases, guanidinobenzotases, sulfatases,lipases, peroxidases, histone deacetylases, exonucleases, reductases,endoglycoceramidases and endonucleases.

In other embodiments, the carrier molecule is an amino acid (includingthose that are protected or are substituted by phosphates,carbohydrates, or C₁ to C₂₂ carboxylic acids), or a polymer of aminoacids such as a peptide or protein. In a related embodiment, the carriermolecule contains at least five amino acids, more preferably 5 to 36amino acids. Such peptides include, but are not limited to,neuropeptides, cytokines, toxins, protease substrates, and proteinkinase substrates. Other peptides may function as organelle localizationpeptides, that is, peptides that serve to target the conjugated compoundfor localization within a particular cellular substructure by cellulartransport mechanisms, including, but not limited to, nuclearlocalization signal sequences. In certain embodiments, the proteincarrier molecules include enzymes, antibodies, lectins, glycoproteins,histones, albumins, lipoproteins, avidin, streptavidin, protein A,protein G, phycobiliproteins and other fluorescent proteins, hormones,toxins and growth factors. In other embodiments, the protein carriermolecule is an antibody, an antibody fragment, avidin, streptavidin, atoxin, a lectin, or a growth factor. In further embodiments, the carriermolecules contain haptens including, but not limited to, biotin,digoxin, digoxigenin and fluorophores.

The carrier molecules used in the methods and composition describedherein can also contain a nucleic acid base, nucleoside, nucleotide or anucleic acid polymer, optionally containing an additional linker orspacer for attachment of a fluorophore or other ligand, such as analkynyl linkage (U.S. Pat. No. 5,047,519), an aminoallyl linkage (U.S.Pat. No. 4,711,955) or other linkage. In other embodiments, thenucleotide carrier molecule is a nucleoside or a deoxynucleoside or adideoxynucleoside, while in other embodiments, the carrier moleculecontains a peptide nucleic acid (PNA) sequence or a locked nucleic acid(LNA) sequence. In certain embodiments, the nucleic acid polymer carriermolecules are single- or multi-stranded, natural or synthetic DNA or RNAoligonucleotides, or DNA/RNA hybrids, or incorporating an unusual linkersuch as morpholine derivatized phosphates (AntiVirals, Inc., CorvallisOreg.), or peptide nucleic acids such as N-(2-aminoethyl)glycine units,where the nucleic acid contains fewer than 50 nucleotides, moretypically fewer than 25 nucleotides.

The carrier molecules used in the methods and composition describedherein can also contain a carbohydrate or polyol, including apolysaccharide, such as dextran, FICOLL, heparin, glycogen, amylopectin,mannan, inulin, starch, agarose and cellulose, or a polymer such as apoly(ethylene glycol). In certain embodiments, the polysaccharidecarrier molecule includes dextran, agarose or FICOLL.

The carrier molecules used in the methods and composition describedherein can also include a lipid including, but not limited to,glycolipids, phospholipids, and sphingolipids. In certain embodiments,such lipids contain 6-25 carbons. In other embodiments, the carriermolecules include a lipid vesicle, such as a liposome,

The carrier molecules used in the methods and composition describedherein can also be a cell, cellular systems, cellular fragment, orsubcellular particles, including virus particles, bacterial particles,virus components, biological cells (such as animal cells, plant cells,bacteria, or yeast), or cellular components. Non-limiting examples ofsuch cellular components that are useful as carrier molecules in themethods and composition described herein include lysosomes, endosomes,cytoplasm, nuclei, histones, mitochondria, Golgi apparatus, endoplasmicreticulum and vacuoles.

The carrier molecules used in the methods and composition describedherein can also non-covalently associates with organic or inorganicmaterials.

The carrier molecules used in the methods and composition describedherein can also include a specific binding pair member wherein thenucleic acid can be conjugated to a specific binding pair member andused in the formation of a bound pair. In certain embodiments, thepresence of a labeled specific binding pair member indicates thelocation of the complementary member of that specific binding pair; eachspecific binding pair member having an area on the surface or in acavity which specifically binds to, and is complementary with, aparticular spatial and polar organization of the other. In certainembodiments, the dye compounds (fluorophores or chromophores) describedherein function as a reporter molecule for the specific binding pair.Exemplary binding pairs are set forth in Table 2.

TABLE 2 Representative Specific Binding Pairs antigen antibody biotinavidin (or streptavidin or anti-biotin) IgG* protein A or protein G drugdrug receptor folate folate binding protein toxin toxin receptorcarbohydrate lectin or carbohydrate receptor peptide peptide receptorprotein protein receptor enzyme substrate enzyme DNA (RNA) cDNA(cRNA)^(†) hormone hormone receptor ion chelator *IgG is animmunoglobulin ^(†)cDNA and cRNA are the complementary strands used forhybridization

In a particular aspect the carrier molecule, used in the methods andcompositions described herein, is an antibody fragment, such as, but notlimited to, anti-Fc, an anti-Fc isotype, anti-J chain, anti-kappa lightchain, anti-lambda light chain, or a single-chain fragment variableprotein; or a non-antibody peptide or protein, such as, for example butnot limited to, soluble Fc receptor, protein G, protein A, protein L,lectins, or a fragment thereof In one aspect the carrier molecule is aFab fragment specific to the Fc portion of the target-binding antibodyor to an isotype of the Fc portion of the target-binding antibody (U.S.Ser. No. 10/118,204). The monovalent Fab fragments are typicallyproduced from either murine monoclonal antibodies or polyclonalantibodies generated in a variety of animals, for example but notlimited to, rabbit or goat. These fragments can be generated from anyisotype such as murine IgM, IgG₁, IgG_(2a), IgG_(2b) or IgG₃.

In alternative embodiments, a non-antibody protein or peptide such asprotein G, or other suitable proteins, can be used alone or coupled withalbumin. Preferred albumins include human and bovine serum albumins orovalbumin. Protein A, G and L are defined to include those proteinsknown to one skilled in the art or derivatives thereof that comprise atleast one binding domain for IgG, i.e. proteins that have affinity forIgG. These proteins can be modified but do not need to be and areconjugated to a reactive moiety in the same manner as the other carriermolecules described.

In another aspect, the carrier molecules, used in the methods andcompositions described herein, can be whole intact antibodies. Antibodyis a term of the art denoting the soluble substance or molecule secretedor produced by an animal in response to an antigen, and which has theparticular property of combining specifically with the antigen thatinduced its formation. Antibodies themselves also serve are antigens orimmunogens because they are glycoproteins and therefore are used togenerate anti-species antibodies. Antibodies, also known asimmunoglobulins, are classified into five distinct classes—IgG, IgA,IgM, IgD, and IgE. The basic IgG immunoglobulin structure consists oftwo identical light polypeptide chains and two identical heavypolypeptide chains (linked together by disulfide bonds).

When IgG is treated with the enzyme papain a monovalent antigen-bindingfragment can be isolated, referred herein to as a Fab fragment. When IgGis treated with pepsin (another proteolytic enzyme), a larger fragmentis produced, F(ab′)₂. This fragment can be split in half by treatingwith a mild reducing buffer that results in the monovalent Fab′fragment. The Fab′ fragment is slightly larger than the Fab and containsone or more free sulfhydryls from the hinge region (which are not foundin the smaller Fab fragment). The term “antibody fragment” is usedherein to define the Fab′, F(ab′)₂ and Fab portions of the antibody. Itis well known in the art to treat antibody molecules with pepsin andpapain in order to produce antibody fragments (Gorevic et al., Methodsof Enzyol., 116:3 (1985)).

The monovalent Fab fragments used as carrier molecules in the methodsand compositions described herein are produced from either murinemonoclonal antibodies or polyclonal antibodies generated in a variety ofanimals that have been immunized with a foreign antibody or fragmentthereof (U.S. Pat. No. 4,196,265 discloses a method of producingmonoclonal antibodies). Typically, secondary antibodies are derived froma polyclonal antibody that has been produced in a rabbit or goat but anyanimal known to one skilled in the art to produce polyclonal antibodiescan be used to generate anti-species antibodies. The term “primaryantibody” describes an antibody that binds directly to the antigen asopposed to a “secondary antibody” that binds to a region of the primaryantibody. Monoclonal antibodies are equal, and in some cases, preferredover polyclonal antibodies provided that the ligand-binding antibody iscompatible with the monoclonal antibodies that are typically producedfrom murine hybridoma cell lines using methods well known to one skilledin the art.

In one aspect the antibodies used as carrier molecules in the methodsand compositions described herein are generated against only the Fcregion of a foreign antibody. Essentially, the animal is immunized withonly the Fc region fragment of a foreign antibody, such as murine. Thepolyclonal antibodies are collected from subsequent bleeds, digestedwith an enzyme, pepsin or papain, to produce monovalent fragments. Thefragments are then affinity purified on a column comprising wholeimmunoglobulin protein that the animal was immunized against or just theFc fragments.

Solid Supports: Azide Reactive, Alkyne Reactive or Phosphine Reactive

In an aspect of the methods and composition described herein, themodified nucleic acids can be covalently conjugated to a solid support.This includes, but is not limited to, any azide modified nucleic aciddisclosed herein and any solid support disclosed herein. In certainembodiments, the modified nucleic acids contain at least one alkynemoiety or at least one phosphine moiety capable of reacting with a solidsupport containing an azide moiety. In other embodiments, the modifiednucleic acids contain at least one azide moiety capable of reacting witha solid support containing an alkyne moiety or a phosphine moiety. Inother embodiments, the modified nucleic acids contain at least onephosphine moiety capable of reacting with a solid support containing anazide moiety. In certain embodiments, the phosphine moieties of themodified nucleic acids and solid supports are triarylphosphine moieties.

A variety of solid supports can be used in the methods and compositionsdescribed herein. Such solid supports are not limited to a specific typeof support, and therefore a large number of supports are available andare known to one of ordinary skill in the art. Such solid supportsinclude, but are not limited to, solid and semi-solid matrixes, such asaerogels and hydrogels, resins, beads, biochips (including thin filmcoated biochips), microfluidic chip, a silicon chip, multi-well plates(also referred to as microtitre plates or microplates), membranes,conducting and nonconducting metals, glass (including microscope slides)and magnetic supports. Other non-limiting examples of solid supportsused in the methods and compositions described herein include silicagels, polymeric membranes, particles, derivatized plastic films,derivatized glass, derivatized silica, glass beads, cotton, plasticbeads, alumina gels, polysaccharides such as Sepharose, poly(acrylate),polystyrene, poly(acrylamide), polyol, agarose, agar, cellulose,dextran, starch, FICOLL, heparin, glycogen, amylopectin, mannan, inulin,nitrocellulose, diazocellulose, polyvinylchloride, polypropylene,polyethylene (including poly(ethylene glycol)), nylon, latex bead,magnetic bead, paramagnetic bead, superparamagnetic bead, starch and thelike. In certain embodiments, the solid supports used in the methods andcompositions described herein are substantially insoluble in liquidphases.

In certain embodiments, the solid support may include a solid supportreactive functional group, including, but not limited to, hydroxyl,carboxyl, amino, thiol, aldehyde, halogen, nitro, cyano, amido, urea,carbonate, carbamate, isocyanate, sulfone, sulfonate, sulfonamide,sulfoxide, wherein such functional groups are used to covalently attachthe azide-containing nucleic acids described herein. In otherembodiments, the solid support may include a solid support reactivefunctional group, including, but not limited to, hydroxyl, carboxyl,amino, thiol, aldehyde, halogen, nitro, cyano, amido, urea, carbonate,carbamate, isocyanate, sulfone, sulfonate, sulfonamide, sulfoxide,wherein such functional groups are used to covalently attach thealkyne-containing nucleic acids described herein. In still otherembodiments, the solid support may include a solid support reactivefunctional group, including, but not limited to, hydroxyl, carboxyl,amino, thiol, aldehyde, halogen, nitro, cyano, amido, urea, carbonate,carbamate, isocyanate, sulfone, sulfonate, sulfonamide, sulfoxide,wherein such functional groups are used to covalently attach thephosphine-containing nucleic acids described herein. In otherembodiments, the solid supports include azide, alkyne or phosphinefunctional groups to covalently attach nucleic acids modified withazide, alkyne or phosphine moieties.

A suitable solid phase support used in the methods and compositionsdescribed herein, can be selected on the basis of desired end use andsuitability for various synthetic protocols. By way of example only,where amide bond formation is desirable to attach the modified nucleicacids described herein to the solid support, resins generally useful inpeptide synthesis may be employed, such as polystyrene (e.g., PAM-resinobtained from Bachem Inc., Peninsula Laboratories, etc.), POLYHIPE™resin (obtained from Aminotech, Canada), polyamide resin (obtained fromPeninsula Laboratories), polystyrene resin grafted with polyethyleneglycol (TentaGel™, Rapp Polymere, Tubingen, Germany),polydimethyl-acrylamide resin (available from Milligen/Biosearch,California), or PEGA beads (obtained from Polymer Laboratories). Incertain embodiments, the modified nucleic acids described herein aredeposited onto a solid support in an array format. In certainembodiments, such deposition is accomplished by direct surface contactbetween the support surface and a delivery mechanism, such as a pin or acapillary, or by ink jet technologies which utilize piezoelectric andother forms of propulsion to transfer liquids from miniature nozzles tosolid surfaces. In the case of contact printing, robotic control systemsand multiplexed printheads allow automated microarray fabrication. Forcontactless deposition by piezoelectric propulsion technologies, roboticsystems also allow for automatic microarray fabrication using eithercontinuous and drop-on-demand devices.

Compositions

In one aspect, the modified nucleic acids, reporter molecules andcarrier molecules provided herein can be used to form a firstcomposition that includes a modified nucleic acids, a first reportermolecule, and a carrier molecule. In another embodiment, a secondnucleic acid that includes a first composition in combination with asecond conjugate, wherein the second conjugate comprises a carriermolecule or solid support that is covalently bonded to a second reportermolecule. The first and second reporter molecules have differentstructures and preferably have different emission spectra. In otherembodiments, the first and second reporter molecules are selected sothat their fluorescence emissions essentially do not overlap. In otherembodiments, the reporter molecules have different excitation spectra,while in other embodiments the reporter molecules have similarexcitation wavelengths and are excited by the same laser. In suchcompositions, the carrier molecule (or solid support) of the conjugatesin the second composition may be the same or a different molecule. Thediscussion herein pertaining to the identity of various carriermolecules is generally applicable to this embodiment as well as otherembodiments.

In another aspect, the modified nucleic acids, reporter molecules andsolid supports provided herein can be used to form a first compositionthat comprises a modified nucleic acid, a first reporter molecule, and asolid support. In another embodiment, a second composition that includesa first composition in combination with a second conjugate. The secondconjugate comprises a solid support or carrier molecule (describedherein) that is covalently bonded to a second reporter molecule. Thefirst and second reporter molecules have different structures andpreferably have different emission spectra. In other embodiments, thefirst and second reporter molecules are selected so that theirfluorescence emissions essentially do not overlap. In other embodiments,the reporter molecules have different excitation spectra, while in otherembodiments the reporter molecules have similar excitation wavelengthsand are excited by the same laser. In such composition, the solidsupport (or carrier molecule) of the conjugates in the secondcomposition may be the same or a different molecule. The discussionherein pertaining to the identity of various solid supports is generallyapplicable to this embodiment of the invention as well as otherembodiments.

Labeling and Separating Modified Nucleic Acids

Methods for forming modified nucleic acid-label (reporter molecule,solid support or carrier molecule) conjugates are described herein. Inone aspect the modified biomolecule-reporter molecule conjugates areformed in solution and then separated using methods known in the art. Itwas unexpectedly found that by adding a copper chelator to the “click”chemistry conjugation reaction the labeling efficiency of modifiednucleic acids and their resolution in gel electrophoresis improved ascompared to those reactions without the addition of a copper chelator.In certain embodiments, the methods of labeling nucleic acids using“click” chemistry, involve an azide modified nucleic acid and a labelthat includes a terminal alkyne that are reacted in a mixture thatincludes copper (II), a reducing agent, and at least one copper (I)chelator. In other embodiments, novel methods are provided for formingconjugates in solution with azide modified nucleic acids and a reportermolecule comprising a terminal alkyne under “click” chemistryconditions. In other embodiments, “click” chemistry is used to formconjugates with alkyne modified nucleic acids and a reporter moleculecomprising an azide. In other embodiments, Staudinger ligation is usedto form conjugates with azide modified nucleic acids and a reportermolecule comprising a phosphine, while other embodiments use Staudingerligation to form conjugates with phosphine modified nucleic acids and areporter molecule comprising an azide. Still other embodiments useactivated alkyne modified nucleic acids to form conjugates with reportermolecules comprising azides, or azide modified nucleic acids formingconjugates with activated alkyne containing reporter molecules.

In other aspects provided herein, the methods of labeling nucleic acidsusing “click” chemistry, wherein a nucleic acid that includes an azidogroup and a label that comprises a terminal alkyne are reacted in amixture that includes copper (II), a reducing agent, and at least onecopper (I) chelator to produce a labeled nucleic acid, results in thepreservation of the structural integrity of the labeled nucleic acid. Inother embodiments, methods of labeling glycoproteins wherein thestructural integrity of the nucleic acid after labeling is not reducedincludes “click” chemistry in which a nucleic acid that includes aterminal alkyne and a label that comprises an azido group are reacted ina mixture that includes copper (II), a reducing agent, and at least onecopper chelator.

The methods for labeling nucleic acids that comprise an azido groupusing “click” chemistry described herein can also be used for nucleicacids that comprise a terminal alkyne, wherein the label to be reactedwith the nucleic acid comprises an azido group. The methods for labelingand detecting nucleic acids that comprise an azido group using “click”chemistry described herein can also be used for nucleic acids thatcomprise a terminal alkyne, wherein the label to be reacted with thenucleic acid comprises an azido group. In one embodiment, is a methodusing the “click” chemistry reaction described herein to form nucleicacid-reporter molecule conjugates in which the reaction mixture includesa reporter molecule with an azide moiety, an alkyne modified nucleicacid, copper (II) ions, at least one reducing agent and a copperchelator. In certain embodiments, such alkyne modified nucleic acids arealkyne modified glycoproteins and such reporter molecule with an azidemoiety are any reporter molecule described herein. In other embodiments,such alkyne modified nucleic acids are alkyne modified glycoproteins andsuch reporter molecule with an azide moiety are any fluorophore basedreporter molecule described herein.

Other methods provided herein, are methods for labeling and detectingseparated nucleic acids using the “click” chemistry cycloadditionreaction described herein. The method includes: combining in a reactionmixture a nucleic acid that comprises an azido group, a label thatincludes a terminal alkyne group, copper (II), a reducing agent, and acopper chelator; incubating the reaction mixture under conditions thatpromote chemical conjugation of the label to the nucleic acid,separating the nucleic acid using one or more biochemical or biophysicalseparation techniques, and detecting the nucleic acid. In otherembodiments, the method includes: combining in a reaction mixture anucleic acid that comprises an alkyne group, a label that includes anazide group, copper (II), a reducing agent, and a copper chelator;incubating the reaction mixture under conditions that promote chemicalconjugation of the label to the nucleic acid, separating the nucleicacid using one or more biochemical or biophysical separation techniques,and detecting the nucleic acid.

In one embodiment is a method for forming a modified nucleic acid label(reporter molecule, solid support or carrier molecule) conjugate,wherein the method includes the steps of:

-   -   a) forming an azide-alkyne cycloaddition reaction mixture that        includes a label having a terminal alkyne moiety, an azido        modified nucleic acid, copper(II) ions, at least one reducing        agent and a copper chelator;    -   b) incubating the azide-alkyne cycloaddition reaction mixture        for a sufficient amount of time to form a nucleic acid-label        conjugate;    -   c) separating the nucleic acid-label conjugate to form a        separated nucleic acid-label conjugate wherein the nucleic acid        label conjugated is formed and separated.

In an alternative embodiment, step a) comprises a label having an azidomoiety and the modified nucleic acid comprises an alkyne.

In another alternative embodiment, step a) comprises forming aStaudinger ligation reaction.

In yet another embodiment, step a) does not comprise copper(II) ions, atleast one reducing agent and a copper chelator wherein the labelcomprises an azido moiety or an activated alkyne and the modifiednucleic acid comprises an azido moiety or an activated alkyne.

In another embodiment is a method for detecting modified nucleic acids,wherein the method includes the steps of:

-   -   a) forming an azide-alkyne cycloaddition reaction mixture that        includes a reporter molecule having a terminal alkyne moiety, an        azido modified nucleic acid, copper(II) ions, at least one        reducing agent and a copper chelator;    -   b) incubating the azide-alkyne cycloaddition reaction mixture        for a sufficient amount of time to form a nucleic acid-reporter        molecule conjugate;    -   c) separating the nucleic acid-reporter molecule conjugate to        form a separated nucleic acid-reporter molecule conjugate;    -   d) illuminating the separated nucleic acid-reporter molecule        conjugate with an appropriate wavelength to form an illuminated        nucleic acid-reporter molecule conjugate, and    -   e) observing the illuminated nucleic acid-reporter molecule        conjugate wherein the nucleic acids is detected.

In another embodiment is a method for detecting modified nucleic acids,wherein the method includes the steps of:

-   -   a) forming an azide-alkyne cycloaddition reaction mixture that        includes a reporter molecule having an azide moiety, an alkyne        modified nucleic acid, copper(II) ions, at least one reducing        agent and a copper chelator;    -   b) incubating the azide-alkyne cycloaddition reaction mixture        for a sufficient amount of time to form a nucleic acid-reporter        molecule conjugate;    -   c) separating the nucleic acid-reporter molecule conjugate to        form a separated nucleic acid-reporter molecule conjugate;    -   d) illuminating the separated nucleic acid-reporter molecule        conjugate with an appropriate wavelength to form an illuminated        nucleic acid-reporter molecule conjugate, and    -   e) observing the illuminated nucleic acid-reporter molecule        conjugate wherein the nucleic acid is detected.

In addition such “click” chemistry reaction mixtures can include,without limitation, one or more buffers, polymers, salts, detergents, orsolubilizing agents. The reaction can be performed under anaerobicconditions, such as under nitrogen or argon gas, and can be performedfor any feasible length of time, such as, for example, from ten minutesto six hours, from about twenty minutes to about three hours, or fromabout thirty minutes to about two hours. The reaction can be performedat a wide range of temperatures, for example ranging from about 4degrees Celsius to about 50 degrees Celsius, and is preferably performedat temperatures between about 10 degrees and about 40 degrees, andtypically between about 15 degrees and about 30 degrees.

Separation and Detection

Another aspect provided herein are methods directed toward detectingmodified nucleic acids after the modified nucleic acids have beenlabeled, using “click” chemistry reactions, Staudinger ligation oractivated alkyne reactions, and separated using, for example,chromatographic methods or electrophoresis methods such as, but notlimited to, gel electrophoresis. In certain embodiments such nucleicacids have been modified using the methods described herein. Theseparation methods used to separate such modified nucleic acidsincludes, but are not limited to, thin layer or column chromatography(including, for example, size exclusion, ion exchange, or affinitychromatography) or isoelectric focusing, gel electrophoresis, capillaryelectrophoresis, capillary gel electrophoresis, and slab gelelectrophoresis. Gel electrophoresis can be denaturing or nondenaturinggel electrophoresis, and can include denaturing gel electrophoresisfollowed by nondenaturing gel electrophoresis (e.g., “2D” gels). Incertain embodiments, the modified nucleic acids are used to formconjugates with a reporter molecule, a carrier molecule and/or a solidsupport prior to separation using the methods described herein. In otherembodiments, the modified nucleic acids are used to form conjugates witha reporter molecule, a carrier molecule and/or a solid support afterseparation using the methods described herein.

In other embodiments, the separation methods used in such separation anddetection methods can be any separation methods used for nucleic acids,such as, for example, chromatography, capture to solid supports, andelectrophoresis. In certain embodiments of such separation and detectionmethods, gel electrophoresis is used to separate nucleic acids and theseparated nucleic acids are detected in the gel by the attached labels.By way of example only, nucleic acids that have incorporated azidomoieties can be labeled in a solution reaction with a terminalalkyne-containing fluorophore, and the nucleic acids can be optionallyfurther purified from the reaction mixture and electrophoresed. Thenucleic acids can be visualized in the gel using light of theappropriate wavelength to stimulate the fluorophore label. Single ordouble stranded nucleic acids can be attached to solid supports prior toincorporation of azido or alkyne nucleotides followed by “click”reaction with a respective azido or alkyne chemical or polymer. Nucleicacids that have alkyne or azide-nucleotides can be attached to solidsupports before or after the click reaction.

Gel electrophoresis can use any feasible buffer system described hereinincluding, but not limited to, Tris-acetate EDTA, Tris-borate EDTA,Tris-glycine, BisTris and Bistris-Tricine. In certain embodiments, theelectrophoresis gel used in the methods described herein compriseacrylamide, including by way for example only, acrylamide at aconcentration from about 2.5% to about 30%, or from about 5% to about20%. In certain embodiments, such polyacrylamide electrophoresis gelcomprise 1% to 10% crosslinker, including but not limited to,bisacrylamide. In certain embodiments, the electrophoresis gel used inthe methods described herein comprises agarose, including by way forexample only, agarose at concentration from about 0.1% to about 5%, orfrom about 0.5% to about 4%, or from about 1% to about 3%. In certainembodiments, the electrophoresis gel used in the methods describedherein comprises acrylamide and agarose, including by way for exampleonly, electrophoresis gels comprising from about 2.5% to about 30%acrylamide and from about 0.1% to about 5% agarose, or from about 5% toabout 20% acrylamide and from about 0.2% to about 2.5% agarose. Incertain embodiments, such polyacrylamide/agarose electrophoresis gelscomprise 1% to 10% crosslinker, including but not limited to,bisacrylamide. In certain embodiments, the gels used to separate nucleicacids can be gradient gels.

The methods described herein can be used to detect modified nucleicacids for “in-gel” detection using slab gel electrophoresis or capillarygel electrophoresis. In one aspect, the method includes combining anazido modified nucleic acid, a label that includes a terminal alkyne,copper (II), a reducing agent, and a copper (I) chelator in a reactionmixture; incubating the reaction mixture under conditions that promotechemical conjugation of the label to the nucleic acid; separating thenucleic acid using one or more biochemical separation techniques; anddetecting the nucleic acid. The label used in such methods can be anylabel described herein. The copper (I) chelator used in such methods canbe any chelator described herein. In certain embodiments, the copper (I)chelator use in such methods is a 1,10 phenanthroline-containing copper(I) chelator. In other embodiments, the copper(I) chelator isbathocuproine disulfonic acid (BCS;2,9-dimethyl-4,7-diphenyl-1,10-phenanthroline disulfonate). In otherembodiments, the copper (I) chelator used in such methods can be used tochelate copper(II).

Without limitation to any specific mechanism, it is known that coppercan promote the cleavage of nucleic acids. The addition of a copperchelator in such methods reduces the detrimental effects of copper usedin the “click” chemistry reactions, and thereby preserves the structuralintegrity of the nucleic acids. Thus, the methods described hereinpreserve the structural integrity of labeled and detected nucleic acids,and thereby provide improved methods of separating and detecting nucleicacids labeled using “click” chemistry. In addition, the methods ofdetecting separated nucleic acids using click chemistry, in which thestructural integrity of the separated molecules is preserved, improvesthe detection of such nucleic acids.

In certain embodiments, the addition of a chelator including, but notlimited to BCS, preserves Telomerase Laddering (see FIG. 10).

In another embodiment of “in-gel” detection, the method includescombining an alkyne modified nucleic acid that comprises a terminalalkyne, a label that includes an azido group, copper (II), a reducingagent, and a copper (I) chelator in a reaction mixture; incubating thereaction mixture under conditions that promote chemical conjugation ofthe label to the nucleic acid; separating the labeled nucleic acid usingone or more biochemical separation techniques; and detecting the nucleicacid. In these methods, the structural integrity of labeled and detectednucleic acids is preserved. The label used in such methods can be anylabel described herein. The copper (I) chelator used in such methods canbe any chelator described herein. In certain embodiments, the copper (I)chelator use in such methods is a 1,10 phenanthroline-containing copper(I) chelator. In other embodiments, the copper(I) chelator isbathocuproine disulfonic acid (BCS;2,9-dimethyl-4,7-diphenyl-1,10-phenanthroline disulfonate. In otherembodiments, the copper (I) chelator used in such methods can be used tochelate copper(II).

In certain embodiments, in-gel fluorescence detection utilizesfluorescent- and/or UV-excitable alkyne containing probes, orfluorescent- and/or UV-excitable azide containing probes. In certainembodiments, the labels used in such separation and detection methodsare any fluorophores described herein which has been derivatized tocontain an alkyne, an azide or a phosphine. In certain embodiments, suchfluorphores include, but are not limited to, fluorescein, rhodamine,TAMRA, an Alexa dye, a SYPRO dye, or a BODIPY dye.

The method described herein can be used for multiplexed detection ofnucleic acids, by labeling a modified nucleic acid using the methodsdescribed herein, and then using a total nucleic acid stain to stain thegel that includes the modified nucleic acids labeled with a fluorophorehaving distinct spectral emission.

In another aspect, nucleic acids can be labeled with an azido tag,electrophoresed on gels, and the resulting gels can be incubated with analkyne tag, such as a fluorescent alkyne tag in the presence of copper(I). Copper (I) can be added in its natural form (e.g. CuBr) or can beproduced in situ from copper (II) compounds with the addition of areducing agent. The reducing agent used in such methods can be anyreducing agent described herein, including but not limited to, ascorbateor TCEP. Addition of a chelator that stabilizes copper (I) can enhancethe chemical ligation. The fluorescent label used in such methods can beany fluorophore described herein. The copper (I) chelator used in suchmethods can be any chelator described herein. In certain embodiments,the copper (I) chelator use in such methods is a 1,10phenanthroline-containing copper (I) chelator. In other embodiments, thecopper(I) chelator is bathocuproine disulfonic acid (BCS;2,9-dimethyl-4,7-diphenyl-1,10-phenanthroline disulfonate). In otherembodiments, the copper (I) chelator used in such methods can be used tochelate copper(II). After the ligation step, the gel is washed and thetagged proteins are visualized using standard fluorescence scanningdevices.

In other embodiments, nucleic acids can be labeled with an alkyne tag,electrophoresed on gels, and the resulting gels can be incubated with anazide tag, such as a fluorescent azide tag in the presence of copper(I). Copper (I) can be added in its natural form (e.g. CuBr) or can beproduced in situ from copper (II) compounds with the addition of areducing agent. The reducing agent used in such methods can be anyreducing agent described herein, including but not limited to, ascorbateor TCEP. Addition of a chelator that stabilizes copper (I) can enhancethe chemical ligation. The fluorescent label used in such methods can beany fluorophore described herein. The copper (I) chelator used in suchmethods can be any chelator described herein. In certain embodiments,the copper (I) chelator use in such methods is a 1,10phenanthroline-containing copper (I) chelator. In other embodiments, thecopper(I) chelator is bathocuproine disulfonic acid (BCS;2,9-dimethyl-4,7-diphenyl-1,10-phenanthroline disulfonate. In otherembodiments, the copper (I) chelator used in such methods can be used tochelate copper(II). After the ligation step, the gel is washed and thetagged proteins are visualized using standard fluorescence scanningdevices.

In further embodiments, nucleic acids can be labeled with an azide tag,electrophoresed on gels, and the resulting gels can be incubated with aphosphine tag, such as a fluorescent phosphine containing tag, usingStaudinger ligation. After the ligation step, the gel is washed and thetagged nucleic acids are visualized using standard fluorescence scanningdevices. In such methods the use of copper, which contributes to thedegradation of nucleic acids such as proteins, can be avoided.

In certain embodiments, a label attached to a nucleic acid using a“click” chemistry reaction with a copper (I) chelator as disclosedherein, can also be used for the separation of nucleic acids. By way ofexample only, affinity chromatography or bead capture techniques can beused to separate nucleic acids labeled with biotin or other affinitytags using the methods described herein. The captured molecules can bedetected using the affinity tags or by other means, and/or furtheranalyzed for structure or function.

Methods for Labeling Immobilized Modified Nucleic Acids

Another aspect provides a method for labeling modified nucleic acidsthat have been immobilized on a solid support. Solid supports used insuch methods have been described herein, and can be solid or semi-solidmatrix. Such solid supports include, but are not limited to, glass,slides, arrays, silica particles, polymeric particles, microtiter platesand polymeric gels. In this aspect the nucleic acids are modified usingthe methods described herein. In certain aspects it is advantageous tofirst immobilize the modified nucleic acids and then to subsequentlyform a nucleic acid conjugate comprising the nucleic acid and a reportermolecule, carrier molecule and the solid support, wherein the reportermolecule, carrier molecule or solid support comprise a reactive groupused to form the conjugate. In certain embodiments such reactive groupsare alkynes for reacting with azides. In certain embodiments suchreactive groups are activated alkynes for reacting with azides. Incertain embodiments such reactive groups are phosphines for reactingwith azides. In certain embodiments such reactive groups are azides forreacting with alkynes. In certain embodiments, the conjugate is formedunder “click” chemistry conditions wherein the reporter molecule,carrier molecule or solid support comprises an alkyne or an azide.

In another aspect the conjugate is formed under Staudinger ligationconditions wherein the reporter molecule, carrier molecule or solidsupport comprises a triaryl phosphine or an azide. In another aspect theconjugate is formed using activated alkynes wherein the reportermolecule, carrier molecule or solid support comprises an activatedalkyne or an azide.

In certain aspects it is advantageous to first immobilize the modifiednucleic acid and then to detect the immobilized nucleic acid usingstandard hybridization techniques wherein the hybridized probe isdetected using methods well known in the art. In this instance themodified nucleic acid comprises a a reactive group that are alkynes forreacting with azides. In certain embodiments such reactive groups areactivated alkynes for reacting with azides. In certain embodiments suchreactive groups are phosphines for reacting with azides. In certainembodiments such reactive groups are azides for reacting with alkynessubsequently form a nucleic acid conjugate comprising the nucleic acidand a solid support, wherein the solid support comprises a reactivegroup used to form the conjugate.

In certain embodiments, it is advantageous to first immobilize the azidomodified nucleic acids and then to subsequently form the nucleic acidconjugate comprising a reporter molecule, carrier molecule or solidsupport wherein the reporter molecule, carrier molecule or solid supportcomprise an azide reactive group prior to forming the conjugate. Incertain embodiments, the conjugate is formed under “click” chemistryconditions wherein the reporter molecule, carrier molecule or solidsupport comprises a terminal alkyne. In another aspect the conjugate isformed under Staudinger ligation conditions wherein the reportermolecule, carrier molecule or solid support comprises a triarylphosphine.

In another aspect, the modified nucleic acid is attached to a solidsupport using functional groups other than functional groups used in“click” chemistry or Staudinger ligation, whereupon the attachedmodified nucleic acid is used to form a conjugate under “click”chemistry conditions or Staudinger ligation with reporter molecules,carrier molecule or another solid support that have functional groupsused in “click” chemistry or Staudinger ligation. By way of exampleonly, the modified nucleic acid can be immobilized to a solid supportusing hydroxyl, carboxyl, amino, thiol, aldehyde, halogen, nitro, cyano,amido, urea, carbonate, carbamate, isocyanate, sulfone, sulfonate,sulfonamide or sulfoxide functional groups. In certain embodiments themodified nucleic acid is an azido modified nucleic acid, an alkynemodified nucleic acid or a phosphine modified nucleic acid.

In certain embodiments, the azido modified nucleic acid is attached to asolid support using functional groups other than azide reactivefunctional groups, whereupon the attached azido modified nucleic acid isused to form a conjugate under click chemistry conditions wherein thereporter molecule, carrier molecule or another solid support comprises aterminal alkyne. In another embodiment the azido modified nucleic acidis attached to a solid support using functional groups other than azidereactive functional groups, whereupon the attached azido modifiednucleic acid is used to form a conjugate under Staudinger ligationconditions wherein the reporter molecule, carrier molecule or othersolid support comprises a triaryl phosphine.

In another aspect is provided a method for detecting immobilized azidomodified nucleic acids, wherein the method includes the following:

-   -   a) immobilizing the azido modified nucleic acids on a solid or        semi-solid matrix to form an immobilized azido modified nucleic        acid;    -   b) contacting the immobilized azido modified nucleic acid with a        reporter molecule that contains an alkyne reactive group to form        a contacted azido modified nucleic acid;    -   c) incubating the contacted azido modified nucleic acid for a        sufficient amount of time to form a reporter molecule-nucleic        acid conjugate;    -   d) illuminating the reporter molecule-nucleic acid conjugate        with an appropriate wavelength to form an illuminated reporter        molecule-nucleic acid conjugate, and    -   e) observing the illuminated reporter molecule-nucleic acid        conjugate whereby the immobilized azido modified nucleic acid is        detected.

In another aspect is provided a method for detecting immobilized alkynemodified nucleic acids, wherein the method includes the following:

-   -   a) immobilizing the alkyne modified nucleic acids on a solid or        semi-solid matrix to form an immobilized alkyne modified nucleic        acid;    -   b) contacting the immbolized alkyne modified nucleic acid with a        reporter molecule that contains an azide reactive group to form        a contacted alkyne modified nucleic acid;    -   c) incubating the contacted alkyne modified nucleic acid for a        sufficient amount of time to form a reporter molecule-nucleic        acid conjugate;    -   d) illuminating the reporter molecule-nucleic acid conjugate        with an appropriate wavelength to form an illuminated reporter        molecule-nucleic acid conjugate, and    -   e) observing the illuminated reporter molecule-nucleic acid        conjugate whereby the immobilized alkyne modified nucleic acid        is detected.

RNAi

RNAi is method for selectively decreasing gene expression and is a costeffective method used to study specific gene targets. Typically RNAioligos are short 20 basepair nucleotides. Hybridization of theoligonucleotide to a targeted gene triggers specific degradation of thegene and thereby decreases gene expression. Using Click modified oligoscould potentially increase the specificity of the Watson-Crick bindingof these short oligonucleotides by increasing the Tm. Similar types ofexperiments have been done with locked nucleic acids (LNA) (Elmen, J. etal 2005, Nuc Acid Res). The Click modification could also potentiallydecrease the susceptibility of the free RNAi oligos to destruction bynucleases after transfection, thereby increasing the half life andeffectiveness of the oligonucleotides.

Methods Using Chemically Labeled Nucleic Acids

It was unexpectedly observed that azide modified-dATP and alkynemodified-dUTP could be incorporated into a nucleic acid polymer usingamplification techniques including, but not limited to PCR. It was alsounexpectedly observed that azide modified-dATP and alkyne modified-dUTPcould be incorporated into a nucleic acid polymer using a Telomeraseresulting in Telomerase laddering (see FIGS. 4-7 (Example 1-4).Telomerase is an enzyme that adds specific DNA sequence repeats(“TTAGGG” in all vertebrates) to the 3′ (“three prime”) end of DNAstrands in the telomere regions, which are found at the ends ofeukaryotic chromosomes. The enzyme is a reverse transcriptase thatcarries its own RNA molecule, which is used as a template when itelongates telomeres, which are shortened after each replication cycle.Therfore, one aspect of the methods of using the modified nucleotidesdescribed herein is in a telomerase activity assay.

In another aspect, such modified nucleotides are labeled usingclick-chemistry based, while in other embodiments such modifiednucleotides are labeled using Staudinger Ligation. In still otherembodiments, such modified nucleotides are labeled using activatedalkyne reactivity. In certain embodiments, the modified nucleotides areazide modified nucleotides which are labeled using click-chemistry,while in other embodiments such modified nucleotides are azidenucleotides labeled using Staudinger Ligation. In still otherembodiments, such modified nucleotides are azide modified nucleotideswhich are labeled using activated alkyne reactivity. In certainembodiments, the modified nucleotides are alkyne modified nucelotideswhich are labeled using click-chemistry, while in other embodiments suchmodified nucleotides are activated alkyne modified nucleotides which arelabeled using activated alkyne reactions with azides. In certainembodiments, the modified nucleotides are azide modified-dATP which arelabeled using click-chemistry (see FIG. 6), while in other embodimentssuch modified nucleotides are azide modified-dATP labeled usingStaudinger Ligation. In still other embodiments, such modifiednucleotides are azide modified-dATP which are labeled using activatedalkyne reactivity. In certain embodiments, the modified nucleotides arealkyne modified-dATP which are labeled using click-chemistry, while inother embodiments such modified nucleotides are activated alkynemodified-dATP which are labeled using activated alkyne reactions withazides. In certain embodiments, the modified nucleotides are azidemodified-dATP which are labeled using click-chemistry, while in otherembodiments such modified nucleotides are azide modified-dUTP labeledusing Staudinger Ligation. In still other embodiments, such modifiednucleotides are azide modified-dATP or dUTP which are labeled usingactivated alkyne reactivity. In certain embodiments, the modifiednucleotides are alkyne modified-dUTP which are labeled usingclick-chemistry (see FIGS. 7-9), while in other embodiments suchmodified nucleotides are activated alkyne modified-dUTP which arelabeled using activated alkyne reactions with azides. The telomeraseassay can serve as a highly significant cancer diagnostic as this enzymeis activated in 90% of known human and other animal cancers. The levelof telomerase activity can be used as a reliable biomarker thatrepresents the different levels of the cancer disease. Therefore, thisassay can help diagnose and evaluate the level of cancer progression ina patient and help determine the response to anticancer treatments.

Thus, in one embodiment is provided a method of measuring TelomeraseEnzyme Activity, comprising steps of:

-   -   a) contacting a cell with an effective amount of a dNTP mix, a        dNTP that comprises an azide group or an alkyne group, a        telomerase substrate primer molecule that may contain a terminal        biotin molecule, a telomerase enzyme such that the azide or        alkyne modified dNTP is incorporated into at least one nucleic        acid polymer;    -   b) contacting the nucleic acid polymer with a reporter molecule        comprising an alkyne, activated alkyne, azide, or phosphine        moiety to form a modified nucleic acid polymer reporter molecule        conjugate;    -   c) separating the modified nucleic acid polymer reporter        molecule from free unreacted reporter; and    -   d) Detecting the labeled nucleic acid reporter molecule        conjugate.

In another aspect, such modified nucleotides described herein areincorporated into nucleic acid polymers using the methods describedherein including, but not limited to, polymerase chain reaction (PCR),ligation-based thermocycling approaches, reverse transcription-PCR,real-time PCR, linear amplification techniques and isothermal DNAamplification techniques such as, by way of example only, real-timestrand displacement amplification (SDA), rolling-circle amplification(RCA), multiple-displacement amplification (MDA), Q-beta replicaseamplification, automated Q-beta replicase amplification assay and otherRNA polymerase mediated techniques such as, for example, nucleic acidsequence based amplification or NASBA. Such incorporated nucleotides arethen labeled using click-chemistry, while in other embodiments suchmodified nucleotides are labeled using Staudinger Ligation. In stillother embodiments, such incorporated nucleotides are labeled usingactivated alkyne reactivity. In certain embodiments, the incorporatednucleotides are azide modified nucleotides which are labeled usingclick-chemistry, while in other embodiments such modified nucleotidesare azide nucleotides labeled using Staudinger Ligation. In still otherembodiments, such incorporated nucleotides are azide modifiednucleotides which are labeled using activated alkyne reactivity. Incertain embodiments, the incorporated nucleotides are alkyne modifiednucelotides which are labeled using click-chemistry, while in otherembodiments such modified nucleotides are activated alkyne modifiednucleotides which are labeled using activated alkyne reactions withazides. In certain embodiments, the incorporated nucleotides are azidemodified-dATP which are labeled using click-chemistry (see FIG. 6),while in other embodiments such modified nucleotides are azidemodified-dATP labeled using Staudinger Ligation. In still otherembodiments, such incorporated nucleotides are azide modified-dATP whichare labeled using activated alkyne reactivity. In certain embodiments,the incorporated nucleotides are alkyne modified-dATP which are labeledusing click-chemistry, while in other embodiments such modifiednucleotides are activated alkyne modified-dATP which are labeled usingactivated alkyne reactions with azides. In certain embodiments, theincorporated nucleotides are azide modified-dUTP which are labeled usingclick-chemistry, while in other embodiments such modified nucleotidesare azide modified-dUTP labeled using Staudinger Ligation. In stillother embodiments, such modified nucleotides are azide modified-dUTPwhich are labeled using activated alkyne reactivity. In certainembodiments, the incorporated nucleotides are alkyne modified-dUTP whichare labeled using click-chemistry (see FIGS. 7-9), while in otherembodiments such modified nucleotides are activated alkyne modified-dUTPwhich are labeled using activated alkyne reactions with azides.

In another aspect, such modified nucleotides described herein areincorporated into nucleic acid polymers using isothermal amplification.Such incorporated nucleotides are then labeled using click-chemistry,while in other embodiments such modified nucleotides are labeled usingStaudinger Ligation. In still other embodiments, such incorporatednucleotides are labeled using activated alkyne reactivity. In certainembodiments, the incorporated nucleotides are azide modified nucleotideswhich are labeled using click-chemistry, while in other embodiments suchmodified nucleotides are azide nucleotides labeled using StaudingerLigation. In still other embodiments, such incorporated nucleotides areazide modified nucleotides which are labeled using activated alkynereactivity. In certain embodiments, the incorporated nucleotides arealkyne modified nucelotides which are labeled using click-chemistry,while in other embodiments such modified nucleotides are activatedalkyne modified nucleotides which are labeled using activated alkynereactions with azides. In certain embodiments, the incorporatednucleotides are azide modified-dATP which are labeled usingclick-chemistry, while in other embodiments such modified nucleotidesare azide modified-dATP labeled using Staudinger Ligation. In stillother embodiments, such incorporated nucleotides are azide modified-dATPwhich are labeled using activated alkyne reactivity. In certainembodiments, the incorporated nucleotides are alkyne modified-dATP whichare labeled using click-chemistry, while in other embodiments suchmodified nucleotides are activated alkyne modified-dATP which arelabeled using activated alkyne reactions with azides. In certainembodiments, the incorporated nucleotides are azide modified-dUTP whichare labeled using click-chemistry, while in other embodiments suchmodified nucleotides are azide modified-dUTP labeled using StaudingerLigation. In still other embodiments, such modified nucleotides areazide modified-dUTP which are labeled using activated alkyne reactivity.In certain embodiments, the incorporated nucleotides are alkynemodified-dUTP which are labeled using click-chemistry (see FIGS. 7-9),while in other embodiments such modified nucleotides are activatedalkyne modified-dUTP which are labeled using activated alkyne reactionswith azides. In particular, FIG. 9 shows that E-dUTP can be incorporatedusing various polymerases, thereby showing an isothermal DNA extensionassay for second strand cDNA synthesis using primer extension.

In certain embodiments, a mixture of modified nucleotides areincorporated using the methods described herein including, but notlimited to, polymerase chain reaction (PCR), ligation-basedthermocycling approaches, reverse transcription-PCR, real-time PCR,linear amplification techniques and isothermal DNA amplificationtechniques such as, by way of example only, real-time stranddisplacement amplification (SDA), rolling-circle amplification (RCA),multiple-displacement amplification (MDA), Q-beta replicaseamplification, automated Q-beta replicase amplification assay and otherRNA polymerase mediated techniques such as, for example, nucleic acidsequence based amplification or NASBA. In certain embodiments themixture of modified nucleotides is a mixture of azide modifiednucleotides, while in other embodiments the mixture of modifiednucleotides is a mixture of alkyne modified nucleotides. In certainembodiments the mixture of modified nucleotides is a mixture of azidemodified dATP and dUTP nucleotides, while in other embodiments themixture of modified nucleotides is a mixture of alkyne modified dATP anddUTP. In certain embodiments, the nucleic acid polymers having amixtures of modified nucleotides is labeled as described above.

FIGS. 4 and 5 demonstrate that azido-dATP is incorporated by telomeraseenzyme which is a reverse transcriptase (RNA dependent DNA polymerase),while FIG. 7 demonstrates that ethynyl-dUTP is also incorporated bytelomerase enzyme. Thus, another aspect of methods using the modifiednucleotides described herein is to detect products of RT-PCR using anucleotide mixtures containing either an ethynyl or azido dNTP orenzymes such as reverse transcriptase and DNA polymerase. The product ofsuch an experiment will be purified and then subjected to click basedlabeling method. The final labeled product can be purified either byprecipitation or size exclusion chromatography. Additionally, thestarting telomerase primer can be biotinylated allowing for purificationof the telomerase modified product after the click reaction. The methodis an example of first strand cDNA synthesis (RT-PCR).

Another aspect using the modified nucleotides described herein is“click” chemistry based oligonucleotide labeling for Fluorescene In-SituHybridization (FISH) and Chromogenic In-Situ Hybridization (CISH) andSilver In-situ Hybridization (SISH). In such methods, standardpolymerases including, but not limited to, Klenow (Exo-), modified orwild type T7 DNA polymerase (Sequenase) or Bst polymerase (Largefragment) are used to amplify a template strand for a given sequenceusing primers as well as using ethynyl or azido dNTPs. The prepared DNAfragments can then be purified and subjected to the “click” reactionwith either azido or alkyne labels, fluorescent labels, Qdots andnanoparticles to create a labeled probe. In certain embodiments, suchprobes are labeled using Staudinger Ligation, while in other embodimentssuch probes are labeled using activated alkyne reactions. Probes formethods like insitu hybridization can also be created using PCR and allof the commercially available PCR polymerases. Isothermal amplificationof plasmids or bacterial artificial chromosomes (BAC) templates usingphi29 DNA polymerase or other polymerases with strand displacementactivity can also be done. In certain embodiments, labeling is doneduring a diagnostic or clinical assay. In other embodiments, suchlabeling in FISH and CISH is an automated in situ hybridizationplatforms where the hybridization can be followed by the click reactionto generate the signal. By way of example only, such automated systemsare instruments from Dako, Ventana Medical Systems, and VisionBiosystems.

In another aspect RNA probes can be used for FISH and CISH, wherein suchprobes are labeled using “click” chemistry. In certain embodiments, suchprobes are labeled using Staudinger Ligation, while in other embodimentssuch probes are labeled using activated alkyne reactions. In certainembodiments, such RNA probes are prepared by the incorporation ofmodified nucleotides using in-vitro transcription system to generate anRNA probe. Alternative methods use small RNA oligonucleotides that canbe labeled via aminoallyl—NHS ester chemistry. In certain embodiments,DNA dependent RNA polymerase from phage T7 or SP6 are used toincorporate alkyne modified oligonucleotide, by way of example onlyethynyl oligonucleotides, or azido oligonucleotides to produce amodified RNA probe. Such modified RNA probes are the labeled with eitherazido or alkyne fluorescent or chromogenic labels using “clickchemistry”, thereby generating fluoregenic or chromogenic RNA probes.

Mofifying Phosphoproteins Using Nucleotide Analogs

In another aspect, phosphoproteins can be modified in vivo or in vitrousing alkyne or azide-tagged nucleotides whereby the azide or alkynemoiety is placed on the gamma phosphate of phosphoroproteins. By way ofexample only, such modifications can be accomplished by adding one ofthe nucleotides shown in FIG. 1 to a reaction mixture containing aprotein kinase and a kinase target molecule. In certain embodiments, thephophoroprotein is an azide-containing phosphoroprotein that can bereacted under “click” chemistry conditions with an alkyne containinglabel including, but not limited to, fluorphores or affinity reagent forquantitation, visualization, or enrichment. In certain embodiments, thephophoroprotein is an alkyne-containing phosphoroprotein that can bereacted under “click” chemistry conditions with an azide containinglabel including, but not limited to, fluorphores or affinity reagent forquantitation, visualization, or enrichment. In other embodiments, suchmodified phosphoroproteins can be used to form conjugates with areporter molecule, a carrier molecule and/or a solid substrate.

In one aspect, modified nucleotide substrates containing azide or alkynemoieties are added directly to cultured cells for metabolicincorporation of the tagged gamma-phosphate molecule into cellularmacromolecules including proteins. The process may involve treatment ofthe cells with pharmacological agents to detect alterations inphosphorylation dynamics. Entry of the compounds into live culturedcells could be enhanced by modifying the nucleotides with functionalgroups that would afford permeability, or by concomitant addition ofcell permeablizing agents.

In another aspect, the kinase reaction could be performed in vitro usingcellular extracts as the source of kinases and substrates. The modifiednucleotides would be added to the reaction mixture and the reactionmixtures incubated with or without the addition of pharmacologicalagents of interest. The in vitro reaction may also entail adding anexogenous kinase or substrate source to the cellular extract along withthe nucleotide analogs. In another application, the method could be usedin vitro without cellular extracts, using purified kinases and kinasesubstrates. In certain embodiments, the kinase reaction can be conductedusing kinase substrates deposited as an array on a solid substrate.

In each of these aspects the reaction mix may contain a buffer optimizedfor the particular kinase(s) of interest, a kinase source, a metal ionsource, glycerol, nucleotide ATP analog, and ATP. The “click” detectionreaction with an alkyne probe would be performed in the presence ofcopper(I), or copper(II) in the presence of a copper(II) reducing agent,a copper(I) chelating agent, and an appropriate buffer to maintaingoptimal pH conditions.

In another aspect of methods using the modified nucleotides describedherein is the preparation of Peptide-nucleic Acid (PNA) Conjugates usingClick Chemistry, Staudinger Ligation or activated alkyne reactions. Insuch methods, a peptide with an O-GlcNac modification on one or moreamino acids is subjected to a GalT1 reaction in the presence ofUDP-GalNAz, resulting in an azido modified peptide. SuchPost-Translational modifications have been described in the co-pendingapplication entitled “Labeling of Glycoproteins” with Ser. No.60/772,221, which is herein incorporated by reference in its entirety.In addition any post-translationally modified protein described in theco-pending application entitled “Labeling of Glcoproteins” with Ser. No.60/772,221 can be used in the methods using modified nucleotide asdescribed herein In certain embodiments, a peptide-nucleic acidconjugate is created by reacting the an azido-linked peptide with analkynyl modified oligonucleotides under “click” chemistry reactionconditions. In certain embodiments, a peptide-nucleic acid conjugate iscreated by reacting the an azido-linked peptide with an alkynyl modifiedoligonucleotides in presence of 1 or 2 mM copper, 10 mM Sodium Ascorbateand 20 mM BCS. In certain embodiments, a peptide-nucleic acid conjugateis created by reacting the an azido-linked peptide with an ethynylmodified oligonucleotides under “click” chemistry reaction conditions.In certain embodiments, a peptide-nucleic acid conjugate is created byreacting the an azido-linked peptide with an ethynyl modifiedoligonucleotides in presence of 1 or 2 mM copper, 10 mM Sodium Ascorbateand 20 mM BCS.

In certain embodiments, a peptide-nucleic acid conjugate is created byreacting the an alkyne-linked peptide with an azide modifiedoligonucleotide under “click” chemistry reaction conditions. In certainembodiments, a peptide-nucleic acid conjugate is created by reacting thean alkyne-linked peptide with an azide modified oligonucleotide inpresence of 1 or 2 mM copper, 10 mM Sodium Ascorbate and 20 mM BCS. Incertain embodiments, a peptide-nucleic acid conjugate is created byreacting an ethynyl-linked peptide with an azide modifiedoligonucleotides under “click” chemistry reaction conditions. In certainembodiments, a peptide-nucleic acid conjugate is created by reacting theethynyl-linked peptide with an azide modified oligonucleotides inpresence of 1 or 2 mM copper, 10 mM Sodium Ascorbate and 20 mM BCS.

Samples and Sample Preparation

The end user will determine the choice of the sample and the way inwhich the sample is prepared. Samples that can be used with the methodsand compositions described herein include, but are not limited to, anybiological derived material or aqueous solution that contains a nucleicacid. In certain embodiments, a sample also includes material in which anucleic acid has been added. The sample that can be used with themethods and compositions described herein can be a biological fluidincluding, but not limited to, whole blood, plasma, serum, nasalsecretions, sputum, saliva, urine, sweat, transdermal exudates,cerebrospinal fluid, or the like. In other embodiments, the samples arebiological fluids that include tissue and cell culture medium whereinnucleic acid of interest has been secreted into the medium. Cells usedin such cultures include, but are not limited to, prokaryotic cells andeukaryotic cells that include primary cultures and immortalized celllines. Such eukaryotic cells include, without limitation, ovary cells,epithelial cells, circulating immune cells, β cells, hepatocytes, andneurons. In certain embodiments, the sample may be whole organs, tissueor cells from an animal, including but not limited to, muscle, eye,skin, gonads, lymph nodes, heart, brain, lung, liver, kidney, spleen,thymus, pancreas, solid tumors, macrophages, mammary glands,mesothelium, and the like.

Various buffers can be used in the methods described herein, includinginorganic and organic buffers. In certain embodiments the organic bufferis a zwitterionic buffer. By way of example only, buffers that can beused in the methods described herein include phosphate buffered saline(PBS), phosphate, succinate, citrate, borate, maleate, cacodylate,N-(2-Acetamido)iminodiacetic acid (ADA), 2-(N-morpholino)-ethanesulfonicacid (MES), N-(2-acetamido)-2-aminoethanesulfonic acid (ACES),piperazine-N,N′-2-ethanesulfonic acid (PIPES),2-(N-morpholino)-2-hydroxypropanesulfonic acid (MOPSO),N,N-bis-(hydroxyethyl)-2-aminoethanesulfonic acid (BES),3-(N-morpholino)-propanesulfonic acid (MOPS),N-tris-(hydroxymethyl)-2-ethanesulfonic acid (TES),N-2-hydroxyethyl-piperazine-N-2-ethanesulfonic acid (HEPES),3-(N-tris-(hydroxymethyl) methylamino)-2-hydroxypropanesulfonic acid(TAPSO), 3-(N,N-Bis[2-hydroxyethyl]amino)-2-hydroxypropanesulfonic acid(DIPSO), N-(2-Hydroxyethyl)piperazine-N′-(2-hydroxypropanesulfonic acid)(HEPPSO), 4-(2-Hydroxyethyl)-1-piperazinepropanesulfonic acid (EPPS),N-[Tris(hydroxymethyl)methyl]glycine (Tricine),N,N-Bis(2-hydroxyethyl)glyeine (Bicine),(2-Hydroxy-1,1-bis(hydroxymethyl)ethyl)amino]-1-propanesulfonic acid(TAPS), N-(1,1-Dimethyl-2-hydroxyethyl)-3-amino-2-hydroxypropanesulfonicacid (AMPSO), tris (hydroxy methyl) amino-methane (Tris),TRIS-Acetate-EDTA (TAE), glycine,bis[2-hydroxyethyl]iminotris[hydroxymethyl]methane (BisTris), orcombinations thereof. In certain embodiments, wherein such buffers areused in gel electrophoresis separations the buffer can also includeethylene diamine tetraacetic acid (EDTA).

The concentration of such buffers used in the methods described hereinis from about 0.1 mM to 1 m. In certain embodiments the concentration isbetween 10 mM to about 1 m. In certain embodiments the concentration isbetween about 20 mM and about 500 mM, and in other embodiments theconcentration is between about 50 mM and about 300 mM. In certainembodiments, the buffer concentration is from about 0.1 mM to about 50mM, while in other embodiments the buffer concentration if from about0.5 mM to about 20 mM.

The pH will vary depending upon the particular assay system, generallywithin a readily determinable range wherein one or more of the sulfonicacid moieties is deprotonated.

In certain embodiments, buffers used in the methods described hereinhave a pH between 5 and 9 at ambient temperature. In certain embodimentsthe buffer has a pH between 6 and 8.5 at ambient temperature. In certainembodiments the buffer has a pH between 6 and 8 at ambient temperature.In certain embodiments the buffer has a pH between 6 and 7 at ambienttemperature. In certain embodiments the buffer has a pH between 5 and 9at 25° C. In certain embodiments the buffer has a pH between 6 and 8.5at 25° C. In certain embodiments the buffer has a pH between 6 and 8 at25° C. In certain embodiments the buffer has a pH between 6 and 7 at 25°C.

In certain embodiments, the samples used in the methods described hereinhave a non-ionic detergent to the sample. Non-limiting examples of suchnon-ionic detergents added to the samples used in the methods describedherein are polyoxyalkylene diols, ethers of fatty alcohols includingalcohol ethoxylates (Neodol from Shell Chemical Company and Tergitolfrom Union Carbide Corporation), alkyl phenol ethoxylates (Igepalsurfactants from General Aniline and Film Corporation), ethyleneoxide/propylene oxide block copolymers (PLURONIC™ Series from BASFWyandotte Corporation), polyoxyethylene ester of a fatty acids (StearoxCD from Monsanto Company), alkyl phenol surfactants (Triton series,including Triton X-100from Rohm and Haas Company), polyoxyethylenemercaptan analogs of alcohol ethoxylates (Nonic 218 and Stearox SK fromMonsanto Company), polyoxyethylene adducts of alkyl amines (Ethoduomeenand Ethomeen surfactants from Armak Company), polyoxyethylene alkylamides, sorbitan esters (such as sorbitan monolaurate) and alcoholphenol ethoxylate (Surfonic from Jefferson Chemical Company, Inc.).Non-limiting examples of sorbitan esters include polyoxyethylene(20)sorbitan monolaurate (TWEEN20), polyoxyethylene(20) sorbitanmonopalmitate (TWEEN40), polyoxyethylene(20) sorbitan monostearate(TWEEN60) and polyoxyethylene(20) sorbitan monooleate (TWEEN 80). Incertain embodiments, the concentration of such non-ionic detergentsadded to a sample is from 0.01 to 0.5%. In other embodiments theconcentration is from about 0.01 to 0.4 vol. %. In other embodiments theconcentration is from about 0.01 to 0.3 vol. %. In other embodiments theconcentration is from about 0.01 to 0.2 vol. %. In other embodiments theconcentration is from about 0.01 to 0.1 vol. %.

Illumination

The compounds and compositions described herein may, at any time before,after or during an assay, be illuminated with a wavelength of light thatresults in a detectable optical response, and observed with a means fordetecting the optical response. In certain embodiments, suchillumination can be by a violet or visible wavelength emission lamp, anarc lamp, a laser, or even sunlight or ordinary room light, wherein thewavelength of such sources overlap the absortion spectrum of afluorpohore or chromaphore of the compounds or compositions decribedherein. In certain embodiments, such illumination can be by a violet orvisible wavelength emission lamp, an arc lamp, a laser, or even sunlightor ordinary room light, wherein the fluorescent compounds, includingthose bound to the complementary specific binding pair member, displayintense visible absorption as well as fluorescence emission.

In certain embodiments, the sources used for illuminating thefluorpohore or chromophore of the compounds or compositions decribedherein include, but are not limited to, hand-held ultraviolet lamps,mercury arc lamps, xenon lamps, argon lasers, laser diodes, blue laserdiodes, and YAG lasers. These illumination sources are optionallyintegrated into laser scanners, flow cytometer, fluorescence microplatereaders, standard or mini fluorometers, or chromatographic detectors.These fluorescence emission of such fluorophores is optionally detectedby visual inspection, or by use of any of the following devices: CCDcameras, video cameras, photographic film, laser scanning devices,fluorometers, photodiodes, photodiode arrays, quantum counters,epifluorescence microscopes, scanning microscopes, flow cytometers,fluorescence microplate readers, or by means for amplifying the signalsuch as photomultiplier tubes. Where the sample is examined using a flowcytometer, a fluorescence microscope or a fluorometer, the instrument isoptionally used to distinguish and discriminate between the fluorescentcompounds of the invention and a second fluorophore with detectablydifferent optical properties, typically by distinguishing thefluorescence response of the fluorescent compounds of the invention fromthat of the second fluorophore. Where a sample is examined using a flowcytometer, examination of the sample optionally includes isolation ofparticles within the sample based on the fluorescence response by usinga sorting device.

In certain embodiments, fluorescence is optionally quenched using eitherphysical or chemical quenching agents. Examples of quenching moietiesinclude, but are not limited to DABCYL (i.e.,4-(4′-dimethylaminophenylazo)-benzoic acid) succinimidyl ester,diarylrhodamine carboxylic acid, succinimidyl ester (or QSY-7), and4′,5′-dinitrofluorescein carboxylic acid, succinimidyl ester (or QSY-33)(all available, for example, from Molecular Probes), quencher1 (Q1;available from Epoch Biosciences, Bothell, Wash.), or “Black holequenchers” BHQ-1, BHQ-2, and BHQ-3 (available from BioSearchTechnologies, Inc., Novato, Calif.).

Kits of the Invention

In another aspect, the present invention provides kits that includeN₃-dATP, an enzyme; an azide reactive reporter molecule, carriermolecule or solid support.

In one aspect, the invention includes a kit for labeling a nucleic acidthat includes at least one label that comprises a terminal alkyne, asolution comprising copper, and a solution that comprises a copper (I)chelator. The kit can further comprise a solution that comprises areducing agent, one or more buffers, or one or more detergents.

In one embodiment, an alkyne label provided in a kit is a fluorophore,such as, but not limited to, a xanthene, coumarin, borapolyazaindacene,pyrene and cyanine In one embodiment, a kit provides two or moredifferent terminal alkyne-containing labels one or more of which is afluorophore, In other embodiments, an alkyne label provided in a kit isa tag, such as but not limited to a peptide or a hapten, such as biotin.

In preferred embodiments, a copper (I) chelator provided in the kit is a1,10 phenanthroline, preferably bathocuproine disulfonic acid. In someembodiments, copper is provided in the form of a copper sulfate orcopper acetate solution. In some embodiments, a reducing agent isprovided in the form of ascorbate.

In another aspect, the invention includes a kit for labeling a nucleicacid that includes at least one label that comprises an azido group, asolution comprising copper, an a solution that comprises a copper (I)chelator. The kit can further comprise a solution that comprises areducing agent, one or more buffers, or one or more detergents.

In one embodiment of this aspect, an azido-containing label provided ina kit is a fluorophore, such as, but not limited to, a xanthene,coumarin, borapolyazaindacene, pyrene and cyanine In other embodiments,an azido label provided in a kit is a tag, such as but not limited to apeptide or a hapten, such as biotin.

In one embodiment, a kit provides two or more different azido-containinglabels one or more of which is a fluorophore, In preferred embodiments,a copper (I) chelator provided in the kit is a 1,10 phenanthroline,preferably bathocuproine disulfonic acid. In some embodiments, copper isprovided in the form of a copper sulfate or copper acetate solution. Insome embodiments, a reducing agent is provided in the form of ascorbate.

A detailed description of the invention having been provided above, thefollowing examples are given for the purpose of illustrating theinvention and shall not be construed as being a limitation on the scopeof the invention or claims.

Examples Example 1 Telomerase Assay with N₃-dUTP and N₃-dATP

TRAPeze™ TELOMERASE assays (Chemicon Kit 57700) were performed withN₃-dUTP and N₃-dATP. Each reaction contained the following:

-   -   (a) 1× TRAP reaction buffer (20 mM Tris-HCl, pH 8.3, 1.5 MgCl₂        63 mM KCl, 0.05% Tween 20, 1 mM EGTA)    -   (b) 50 μM of different combinations of deoxynucleoside        triphosphates (see Table 1 for details)    -   (c) 1 μl of TS primer (TRAPeze Telomerase Kit, Chemicon)    -   (d) 1 μl of Primer Mix (contains three separate primers—a K1 Fwd        primer, RP Rev Primer and TSK 1 internal control primer from the        (TRAPeze Telomerase Kit, Chemicon)    -   (e) 2 units of Taq DNA polymerase    -   (f) 2 μl of positive control cells (500 cells) of the TRAPeze        Telomerase Kit

(Chemicon). The cell extract was prepared and further diluted in CHAPSbuffer, the components of which were 10 mM Tris-HCl pH 7.5, 1 mM MgCl₂,1 mM EGTA, 0.1 mM Benzamidine, 5 mm β-mercaptoethanol, 0.5% CHAPS and10% Glycerol.

-   -   (g) PCR grade water as required to bring the volume of each        reaction to 50 μl.

The reaction was carried out in an Applied Biosystems PCR instrumentunder the following conditions: 30° C.-30 minutes-hold

-   -   32 cycles of 95° C.-30 sec        -   59° C.-30 sec        -   72° C.-1 minute    -   Followed by: 4° C.-infinity

The reactions were pulled out, mixed with TRACK-IT Cyan orange loadingdye. The reactions labeled 1,2,3,4 (see FIG. 4 and Table 1) weresubjected to a 4-20% Tri Borate EDTA—polyacrylamide gel. The gel was runat 10V for 10 minutes; 190V for 90 minutes. The gel was then pulled outof the cassette and stained with 1:10,000 fold dilution of stock SYBRGOLD in TBE for 20-30 minutes and then scanned for a signal (Ex: 473,Em:520).

The results of the amplification are shown in FIG. 4, and the listing ofthe dNTP combinations in each reaction (gel lane) is given in Table 1.

TABLE 1 dNTP combinations in the reactions Lanes (in gel) dNTPs 1 50 μMof dATP dTTP, dCTP, and dGTP. 2 50 μM of N₃-dUTP, dATP, dCTP, and dGTP 350 μM of N₃-dATP, dATP, dCTP, and dGTP 4 50 μM of N₃-dUTP, N₃-dATP,dCTP, and dGTPThe left most lane labeled “L” on the gel is a 10 bp ss DNA ladder (200ng per lane).

From FIG. 4 it is seen that N₃-dUTP is incorporated by Taq polymerase(lane 2), and that N₃-dATP is incorporated both by telomerase and Taqpolymerase (lane 3).

Example 2 Dose-Dependence of Telomerase Assay with N₃-dUTP and N₃-dATP

TRAPeze™ TELOMERASE assays (Chemicon Kit 57700) were performed withN₃-dUTP and N₃-dATP. Each reaction contained the following:

-   -   (a) 1× TRAP reaction buffer (20 mM Tris-HCl, pH 8.3, 1.5 MgCl₂        63 mM KCl, 0.05% Tween 20, 1 mM EGTA)    -   (b) 50 μM of deoxynucleoside triphosphates (see Table 2 for        details)    -   (c) 1 μl of TS primer (TRAPeze Telomerase Kit, Chemicon)    -   (d) 1 μl of Primer Mix (contains three separate primers—a K1 Fwd        primer, RP Rev Primer and TSK 1 internal control primer from the        (TRAPeze Telomerase Kit, Chemicon)    -   (e) 2 units of Taq DNA polymerase    -   (f) 2 μl of positive control cells (500 cells) of the TRAPeze        Telomerase Kit

(Chemicon). The cell extract was prepared and further diluted in CHAPSbuffer, the components of which were 10 mM Tris-HCl pH 7.5, 1 mM MgCl₂,1 mM EGTA, 0.1 mM Benzamidine, 5 mm β-mercaptoethanol, 0.5% CHAPS, and10% Glycerol

-   -   (g) PCR grade water as required to bring the volume of each        reaction to 50 μl.

The reaction was carried out in an Applied Biosystems PCR instrumentunder the following conditions: 30° C.-30 minutes-hold (At the end ofthe 30 minute Telomerase reaction, appropriate volumes of azido-dNTPswere added to bring the final concentration to 50 μM. The PCR step wasthen carried out after that)

-   -   32 cycles of 95° C.-30 sec        -   59° C.-30 sec        -   72° C.-1 minute    -   Followed by: 4° C.-infinity

The reactions were pulled out, mixed with TRACK-IT Cyan orange loadingdye. The reactions labeled 1,2,3,4 (see FIG. 5 and Table 2) weresubjected to a 4-20% Tris Borate EDTA—polyacrylamide gel. The gel wasrun at 10V for 10 minutes; 190V for 90 minutes. The gel was then pulledout of the cassette and stained with 1:10,000 fold dilution of stockSYBR GOLD in TBE for 20-30 minutes and then scanned for a signal (Ex:473, Em:520).

The results of the amplification are shown in FIG. 5, and the listing ofthe dNTP combinations in each reaction (gel lane) is given in Table 2.

TABLE 2 dNTP combinations in the reactions Lanes (in gel) dNTPs 1 50 μMof dATP, dTTP, dCTP, and dGTP. 2 50 μM N₃-dUTP, dATP, dCTP, and dGTP 310 μM N₃-dUTP, 50 μM dATP, dCTP, and dGTP 4 1 μM N₃-dUTP, 50 μM dATP,dCTP, and dGTP 5 0.1 μM N₃-dUTP, 50 μM dATP, dCTP, and dGTP 6 50 μMN₃-dATP, dTTP, dCTP, and dGTP 7 10 μM N₃-dATP, 50 μM dATP, dCTP, anddGTP 8 1 μM N₃-dATP, 50 μM dATP, dCTP, and dGTP 9 0.1 μM N₃-dATP, 50 μMdATP, dCTP, and dGTPThe left most lane labeled “L” on the gel is a 10 bp ss DNA ladder (200ng per lane).

From FIG. 5 it is seen that N₃-dUTP is not incorporated by theTelomerase enzyme within a range of 100 nM to 50 micro molar, but it isincorporated by Taq polymerase (lane 2-5). However, N₃-dATP isincorporated both by telomerase and Taq polymerase (lanes 6-9).

Example 3 “Click”-Chemistry Based Telomerase Activity AssayDose-Dependence of Telomerase Assay with N₃-dATP

TRAPeze™ TELOMERASE assays (Chemicon Kit 57700) were performed withN₃-dATP. Each reaction contained the following:

-   -   (a) 1× TRAP reaction buffer (20 mM Tris-HCl, pH 8.3, 1.5 MgCl₂        63 mM KCl, 0.05% Tween 20, 1 mM EGTA)    -   (b) 50 μM of different combinations of deoxynucleoside        triphosphates (see Table 3 for details)    -   (c) 1μl of TS primer (TRAPeze Telomerase Kit, Chemicon)    -   (d) 1μl of Primer Mix (contains three separate primers—a K1 Fwd        primer, RP Rev Primer and TSK 1 internal control primer from the        (TRAPeze Telomerase Kit, Chemicon)    -   (e) 2 units of Taq DNA polymerase    -   (f) 2 μl of positive control cells (500 cells) of the TRAPeze        Telomerase Kit (Chemicon). The cell extract was prepared and        further diluted in CHAPS buffer, the components of which were 10        mM Tris-HCl pH 7.5, 1 mM MgCl₂, 1 mM EGTA, 0.1 mM Benzamidine, 5        mm β-mercaptoethanol, 0.5% CHAPS, and 10% Glycerol    -   (g) PCR grade water as required to bring the volume of each        reaction to 50 μl.

The reaction was carried out in an Applied Biosystems PCR instrumentunder the following conditions: 30° C.-30 minutes-hold

-   -   32 cycles of 95° C.-30 sec        -   59° C.-30 sec        -   72° C.-1 minute    -   Followed by: 4° C.-infinity

The reactions were cleaned through size exclusion columns (ChromaspinTE30, Clonetech). The eluate was then subjected to click reaction usinga final concentration of 25% propylene glycol; 1 mM copper sulfate; 10mM bathocuproinedisulfonic acid (BCS), 10 mM Sodium Ascorbate and 50 μMalkyne-TAMRA. The reaction was performed for 30 minutes at roomtemperature. This was followed by clean up on a size exclusion asdescribed above. The reactions were pulled out, mixed with the TRACK-ITCyan orange loading dye. The reactions (see FIG. 6 and Table 3) weresubjected to a 20% TBE—polyacrylamide gel. The gel was run at 10V for 10minutes; 190V for 90 minutes. The gel was pulled out of the cassette andscanned for TAMRA (Ex: 530 nm Em: 580 nm). The same gel was then stainedwith SYBR GOLD for 30 minutes and then scanned as described above.

The results of the amplification are shown in FIG. 6, and the listing ofthe dNTP combinations in each reaction (gel lane) is given in Table 3.

TABLE 3 dNTP combinations in the reactions Lanes (in gel) dNTPs 1 or a50 μM of dATP, dTTP, dCTP, and dGTP. 2 or b 50 μM of dATP, dTTP, dCTP,and dGTP 3 or c 10 μM N₃-dATP + 40 μM dATP, 50 μM dATP, dCTP, and dGTP 4or d 30 μM dATP + 20 μM N₃-dATP, 50 μM dTTP, dCTP, and dGTP 5 or e 25 μMdATP + 25 μM N₃-dATP, 50 μM dTTP, dCTP, and dGTP 6 or f 20 μM dATP + 30μM N₃-dATP, 50 μM dTTP, dCTP, and dGTP 7 or g 10 μM dATP + 40 μMN₃-dATP, 50 μM dTTP, dCTP, and dGTP 8 or h 50 μM N₃-dATP, 50 μM dTTP,dCTP, and dGTPThe left most lane labeled “L” on the gel is a 10 bp ss DNA ladder (200ng per lane).

From FIG. 6 it is seen that there is a dose dependence of the “click”reaction with the best signal coming from the 100% 50 μM of N₃-dATP.Also the PCR reaction with 50 μM of N₃-dATP was not as efficient.

Example 4 “Click”-Chemistry Based Telomerase Activity AssayDose-Dependence of Telomerase Assay with E-dUTP

TRAPeze™ TELOMERASE assays (Chemicon Kit 57700) were performed withethynyl-dUTP. Each reaction contained the following:

-   -   (a) 1× TRAP reaction buffer (20 mM Tris-HCl, pH 8.3, 1.5 MgCl₂        63 mM KCl, 0.05% Tween 20, 1 mM EGTA)    -   (b) 50 μM of different combinations of deoxynucleoside        triphosphates (see Table 4 for details)    -   (c) 1 μl of TS primer (TRAPeze Telomerase Kit, Chemicon)    -   (d) 1 μl of Primer Mix (contains three separate primers—a K1 Fwd        primer, RP Rev Primer and TSK 1 internal control primer from the        (TRAPeze Telomerase Kit, Chemicon)    -   (e) 2 units of Taq DNA polymerase    -   (f) 2 μl of positive control cells (500 cells) of the TRAPeze        Telomerase Kit (Chemicon). The cell extract was prepared and        further diluted in CHAPS buffer, the components of which were 10        mM Tris-HCl pH 7.5, 1 mM MgCl₂, 1 mM EGTA, 0.1 mM Benzamidine, 5        mm β-mercaptoethanol, 0.5% CHAPS, and 10% Glycerol    -   (g) PCR grade water as required to bring the volume of each        reaction to 50 μl.

The reaction was carried out in an Applied Biosystems PCR instrumentunder the following conditions: 30° C.-30 minutes-hold

-   -   32 cycles of 95° C.-30 sec        -   59° C.-30 sec        -   72° C.-1 minute    -   Followed by: 4° C.-infinity

The reactions were cleaned through size exclusion columns (ChromaspinTE30, Clonetech). The eluate was then subjected to click reaction usinga final concentration of 25% propylene glycol; 1 mM copper sulfate; 10mM BCS, 10 mM Sodium Ascorbate and 50 μM azido-TAMRA. The reaction wasperformed for 30 minutes at room temperature. This was followed by cleanup on a size exclusion as described above. The reactions were pulledout, mixed with the TRACK-IT Cyan orange loading dye. The reactions (seeFIG. 7 and Table 4) were subjected to a 20% TBE—polyacrylamide gel. Thegel was run at 10V for 10 minutes; 190V for 90 minutes. The gel waspulled out of the cassette and scanned for TAMRA (Ex: 530 nm Em: 580nm). The same gel was then stained with SYBR GOLD for 30 minutes andthen scanned as described above.

The results of the amplification are shown in FIG. 7, and the listing ofthe dNTP combinations in each reaction (gel lane) is given in Table 4.

TABLE 4 dNTP combinations in the reactions Lanes (in gel) dNTPs 1 or A50 μM of dATP, dTTP, dCTP, and dGTP. 2 or B 50 μM of dATP, dTTP, dCTP,and dGTP 3 or C 40 μM dTTP + 10 μM e-dUTP, 50 μM dATP, dCTP, and dGTP 4or D 30 μM dTTP + 20 μM e-dUTP, 50 μM dATP, dCTP, and dGTP 5 or E 25 μMdTTP + 25 μM e-dUTP, 50 μM dATP, dCTP, and dGTP 6 or F 20 μM dTTP + 30μM e-dUTP, 50 μM dATP, dCTP, and dGTP 7 or G 10 μM dTTP + 40 μM e-dUTP,50 μM dATP, dCTP, and dGTP 8 or H 50 μM e-dUTP, 50 μM dTTP, dCTP, anddGTPThe left most lane labeled “L” on the gel is the 10 bp ss DNA ladder(200 ng per lane).

From FIG. 7 it is seen that there is a dose dependence of the clickreaction with the best signal coming from the 100% 50 μM of N₃-dATP.Also the PCR reaction with 50 μM of E-dUTP was as efficient as withnatural dNTPs.

Example 5 PCR Incorporation and Detection of Azide or Alkyne NucleicAcids

The reaction was carried out in an Applied Biosystems PCR instrumentunder the following conditions: 30° C.-30 minutes-hold

-   -   32 cycles of 95° C.-30 sec        -   59° C.-30 sec        -   72° C.-1 minute    -   Followed by: 4° C.-infinity        In addition, PCR was preformed with Taq and Pfu-turbo DNA        polymerase. Briefly, a 1 pmol of either a 36 (lane 1/5/1/f) or        38 (lanes 2/6/b/g) or 44 (lanes 3/7/c/h)or 60 bp (lanes 4/8/d/1)        amplicon was amplified by PCR using 10 nmolar forward and        reverse Primer, 50 μM modified dNTPs (e-dUTP, dTTP, dCTP, and        dGTP), 1× Taq or Pfu Turbo buffer and 1.5 mM MgCl2 (for Taq        polymerase).

The reactions were cleaned through size exclusion columns (ChromaspinTE30, Clonetech). The eluate was then subjected to click reaction usinga final concentration of 25% propylene glycol; 1 mM copper sulfate; 10mM BCS, 10 mM Sodium Ascorbate and 50 μM azido-TAMRA or 50 μMalkyne-TAMRA. The reaction was performed for 30 minutes at roomtemperature. This was followed by clean up on a size exclusion asdescribed above. The reactions were pulled out, mixed with the TRACK-ITCyan orange loading dye. The reactions (see FIG. 8 and Table 4) weresubjected to a 20% TBE—polyacrylamide gel. The gel was run at 10V for 10minutes; 190V for 90 minutes. The gel was pulled out of the cassette andscanned for TAMRA (Ex: 530 nm Em: 580 nm). The same gel was then stainedwith SYBR GOLD for 30 minutes and then scanned as described above.

FIG. 8 shows a 20% TBE PAGE that has been scanned for TAMRA (left; Ex532-Em 580) followed by staining with SYBR GOLD (right). Lanes 1, 2, 3and 4 (or a, b, c, d) have been loaded with 2 ul of the PCR productwhile lanes 5,6,7 and 8 (or f, g, h, 1, k) have been loaded with 6 ul ofthe PCR product. The fact that one can see ladders or the 36 bp internalcontrol band in a TRAP assay indicates that not only can Telomeraseincorporate azido or alkyne modified compounds but Taq polymerase canalso do the same, since the subsequent step in TRAP assay afterTelomerase reaction is the PCR step.

Example 6 Incorporation of E-dUTP in an Isothermal Extension Assay UsingDifferent Polymerases

Single stranded DNA oligomers shown below were used for a primerextension assay using “clickable” dNTPs. The oligos used were designedto titrate the dUTP in the sequence.

Oligo 3

(SEQ ID NO: 1) 5′-TTAGGGTTAGGGTTAGGGTTTGGGTTTGGGTTTGGGTTTGGGTTTGGGTTTGGGCTGGCCGTCGTTTTAC

Oligo 4

(SEQ ID NO: 2) 5′-TTTGGGTTTGGGTTTGGGTTTGGGTTTGGGTTTGGGTTAGGGTTTGGGTTTGGGCTGGCCGTCGTTTTACM13 Primer for Annealing at the End of all of these Oligos

5′-GTAAAACGACGGCCAG-3′ (SEQ ID NO: 3)

100 pmol of Oligo 3 or 4 were annealed with the 500 pmol M13 primer inan annealing buffer (7 mM Tris-HCl pH 7.5, 2.5 mM MgCl2, 20 mM NaCl).The reaction mix was heated up to 95 C for 5 minutes, 20 minutes at 65 Cand then cooled to ambient temperatures. All the reactions were suppliedwith 50 uM dNTPs each. The dNTP mix consisted of 50 uM of e-dUTP, dATP,dGTP, dCTP each. The reaction was initiated by addition of thepolymerase. Details of the different reactions are given in Table 5:

TABLE 5 Lane Oligo polymerase Conditions of experiment 1 or a O3 Klenow(Exo −ve) Annealing buffer/30 ‘ @37 C. 2 or b O3 Taq polymerase 1X TSbuffer (1.5 mM MgCl2)/ 30 ‘ @72 C. for 30, in a dry bath 3 or c O3 Pfu(Turbo) 1 X Pfu turbo buffer/95 C. for 30 seconds; 59 C. for 1 minute;72 C. for 2’ in an applied biosystems PCR instrument 4 or d O3 Taqpolymerase 1 X annealing buffer (2.5 mM MgCl2)/same conditions as forreaction 3 5 or e O4 Klenow (Exo −ve) Annealing buffer/30 ‘ @37 C. 6 orf O4 Taq polymerase 1X TS buffer (1.5 mM MgCl2)/ 30 ‘ @72 C. for 30, ina dry bath 7 or g O4 Pfu (Turbo) 1 X Pfu turbo buffer/95 C. for 30seconds; 59 C. for 1 minute; 72 C. for 2’ in an applied biosystems PCRinstrument 8 or h O4 Pfu (Turbo) 1 X Pfu turbo buffer/95 C. for 30seconds; 59 C. for 1 minute; 72 C. for 2’ in an applied biosystems PCRinstrumentLanes designated L_(a) and L _(b) are the 25 bp DNA ladder.

The extended dsDNA product was subjected to a “click” reaction. Thevolume of the reaction product was brought to 25 of 50 ul. The finalconcentrations of the reaction components were 25% propylene glycol, 1mM Copper (II), 10 mM Sodium Ascorbate, 10 mM BCS and 50 uM azido-TAMRA.The reactions were rocked on a tube shaker for 30-60 minutes at roomtemperature. The contents of the tube were then subjected to sizeexclusion chromatography using Chroamspin columns. The purified ds DNAwas then mixed with 1/10 volume of 10× blue juice and loaded on to a 2-%TBE PAGE which was run at constant 200V for 2 hours.

After completion of the run the gel was scanned for TAMRA (Ex: 530 nmand Em 580 nm), the results of which are shown on the left part of FIG.9. After scanning for TAMRA the gel was stained with 1:10,000 folddilution of SYBR GOLD in 1× TBE and scanned for signal (Ex: 473 Em:580)shown on the right.

Example 7 Incorporation of Cu(I) Chelator BCS to Preserve TrAP Laddering

TRAPeze™ TELOMERASE assays (Chemicon Kit 57700) were performed with BCS.Each reaction contained the following:

-   -   (a) 1× TRAP reaction buffer (20 mM Tris-HCl, pH 8.3, 1.5 MgCl₂        63 mM KCl, 0.05% Tween 20, 1 mM EGTA)    -   (b) 50 of N₃-dATP+50 μM of dGTP, dCTP, dTTP. (see Table 6 for        details)    -   (c) 1 μl of TS primer (TRAPeze Telomerase Kit, Chemicon)    -   (d) 1 μl of Primer Mix (contains three separate primers—a K1 Fwd        primer, RP Rev Primer and TSK 1 internal control primer from the        (TRAPeze Telomerase Kit, Chemicon)    -   (e) 2 units of Taq DNA polymerase    -   (f) 2 μl of positive control cells (500 cells) of the TRAPeze        Telomerase Kit

(Chemicon). The cell extract was prepared and further diluted in CHAPSbuffer, the components of which were 10 mM Tris-HCl pH 7.5, 1 mM MgCl₂,1 mM EGTA, 0.1 mM Benzamidine, 5 mm β-mercaptoethanol, 0.5% CHAPS and10% Glycerol.

(g) PCR grade water as required to bring the volume of each reaction to50 μl.

The reaction was carried out in an Applied Biosystems PCR instrumentunder the following conditions: 30° C.-30 minutes-hold

-   -   32 cycles of 95° C.-30 sec        -   59° C.-30 sec        -   72° C.-1 minute    -   Followed by: 4° C.-infinity

The reactions were cleaned through size exclusion columns (ChromaspinTE30, Clonetech). The eluate was then subjected to click reaction usinga final concentration of 25% propylene glycol; 1 mM copper sulfate; inpresence or absence of 10 mM BCS (see Table 6), 10 mM Sodium Ascorbateand 50 μM alkyne-TAMRA. The reaction was performed for 30 minutes atroom temperature. This was followed by clean up on the size exclusion asdescribed above. The reactions were mixed with the TRACK-IT Cyan orangeloading dye and were subjected to a 20% TBE—polyacrylamide gel (see FIG.10). The gel was run at 10V for 10 minutes; 190V for 90 minutes. The gelwas pulled out of the cassette and scanned for TAMRA (Ex: 530 nm Em: 580nm). The same gel was then stained with SYBR GOLD for 30 minutes andthen scanned with an excitation source at 473 nm and emission at 520 nm.

TABLE 6 Chelator for Lane Telomerase click labeling Comments 1 or a 500cells none All other components (Telomerase +ve) of the click reaction 2or b 500 cells 10 mM BCS are as described (Telomerase +ve) 3 or c 500cells None Sau 3 cells do not express (Telomerase −ve) Telomerase. 4 ord 500 cells 10 mM BCS (Telomerase −ve) 5 or e Heat inactivated NoneTelomerase enzyme is cells sensitive to heat. Heating 6 or f Heatinactivated 10 mM BCS the +ve control cells cells for 10 minutes at 80C. destroys telomerase activity.

The results of the amplification are shown in FIG. 10, where the gelshows aa high molecular weight product that appears in the absence ofBCS irrespective of the activity of Telomerase. However, theTRAP“laddering” pattern of the assay product is restored upon additionof the BCS.

Example 8 Click Chemistry Based Detection of Amplified DNA ProductsUsing Isothermal DNA Amplification Technology-Helicase DependentAmplification (See FIG. 11)

8a) Helicase Dependent amplification (see FIG. 11)

a. mHDA: A mixture of DNA Helicase, a DNA polymerase,deoxyoligonucleotide primers and deoxynucleotide triphosphates witheither: (1) an azido-dATP or an ethylene-dUTP (in place of dTTP) or (2)an azido-dUTP or ethylene-dUTP in addition to the four dNTPs are addedtogether. The reaction mixture is heated to 95° C. for 5 minutesfollowed by incubation of the reaction mix at 37° C. for 1 to 3 hoursdepending upon the length of the target and amount of final productrequired.

After completion the polymerase reaction is complete, Click reactioncomponents are added to the amplified DNA. The click reaction componentsare CuSo4, BCS, Na-Ascorbate and either an azido-fluorophore or analkyne-flurophore. The reaction is then run directly on an agarose gelor detected using a secondary matrix.

8b) b. tHDA: A mixture of DNA Helicase, Bst DNA polymerase (fromBacillus stearoethermophillus), deoxyoligonucleotide primers anddeoxynucleotide triphosphates with either: (1) an azido-dATP or anethylene-dUTP (in place of dTTP) or (2) an azido-dUTP or ethylene-dUTPin addition to the four dNTPs are added together. The reaction mixtureis heated to 95° C. for 5 minutes followed by incubation of the reactionmix at 65° C. for 1 to 2 hours depending upon the length of the targetand amount of final product required.

After completion the polymerase reaction is complete, Click reactioncomponents are added to the amplified DNA. The click reaction componentsare CuSO₄, BCS, Na-Ascorbate and either an azido-fluorophore or analkyne-flurophore. The reaction is then run directly on an agarose gel.

c. Circular HDA: This method of DNA amplification uses T7 Helicase andT7DNA polymerase and is similar to rolling circle DNA amplification.Other accessory proteins in this platform include T7 single strand DNAbinding protein. This platform can be used for invitro amplification ofplasmid or covalent closed circular DNA. This technology has significantuse in clinical diagnostics and molecular biology e.g., in DNAsequencing and mutagenesis. As described above azido or alkyne modifiednucleotides triphosphates are used during the DNA amplification methodsand then either the alkyne or azido dye molecules are added to create alabel on the newly synthesized DNA.

d. rt-HDA: this method takes advantage of the reverse transcriptaseactivity of reverse transcriptase under constant temperature conditionscombined with polymerase activity of Bst polymerase. Detection of theamplified DNA is performed as described above using an azido or alkynedNTPs and azido/alkyne dyes are added at the end of the amplificationreaction under conditions that promote a Click reaction between themodified dNTP and the dye label.

Strand Displacement Amplification (SDA) (see FIG. 12):

SDA is an isothermal nucleic acid amplification method. Primercontaining a restriction site is annealed to template. Amplificationprimers are then annealed to 5′ adjacent sequences (form a nick) andamplification is started at a fixed temperature. Newly synthesized DNAare nicked by a restriction enzyme, polymerase starts amplificationagain, displacing the newly synthesized strands. One hundred and ninecopies of DNA can be made in one reaction. For better labeling anddetection of the amplified product, an azido or alkyne dUTP is addedwhich will be incorporated into the newly synthesized strand because theenzyme is a member of a family of pol I DNA polymerases which have beenshown in the art to incorporate azido or alkyne modified dNTPs using Taqpolymerase. Once the amplification reaction is finished, the azido oralkyne dNTP in the polymerized strand is ligated to an azido or alkynedye under conditions that will promote the Click reaction.

Loop mediated Isothermal DNA amplification:

LAMP (Loop-mediated Isothermal Amplification) method is a nucleic acidamplification method that uses 4 primers, which recognize 6 distinctregions on the target gene and a DNA polymerase with strand displacementactivity to carry out reaction under isothermal condition. Amplificationand detection of a gene can be completed in a single step, by incubatingthe mixture of samples, primers, DNA polymerase with strand displacementactivity and substrate at a constant temperature between 60-65° C. Themethod provides high amplification efficiency, with DNA being amplified109-110 times in 15-60 minutes. Because of its high specificity, thepresence of amplified product can indicate the presence of target gene.Since this also uses Bst DNA polymerase, Click chemistry can be used todetect labeling.

Rolling circle DNA amplification/Phi29 based DNA amplification:

This method uses phi 29 DNA polymerase and can amplify DNA (Linear orcircular) with high fidelity and efficiency. Many labs, industrial andacademic use it for clinical pathology, academic research andpreparation of DNA probes from in situ hybridizations. We propose to doeither of the following steps:

(1) add either an azido-dUTP or ethylene-dUTP to replace dTTP in thereaction mix or

(2) add either an azido-dUTP or ethylene-dUTP in addition to the fourdNTPs

Once the polymerase reaction is complete, Click reaction components areadded to the amplified DNA. The Click reaction components are CuSo₄,BCS, Na-Ascorbate and either an azido-fluorophore or analkyne-flurophore. The reaction can then be run directly on an agarosegel

As has been described above, Click chemistry can be used betweenazido/alkyne nucleotides and alkyne/azido dyes to label and detect DNAin other isothermal DNA amplification technologies such as multipledisplacement amplification, transcription mediated amplification, etc.

Preparation of probes for ISH/FISH

The probes for in situ hybridization can also be made using “Click”labeling. Using standard polymerases e.g., Klenow (Exo-), T7 DNApolymerase (Sequenase) or Bst polymerase (Large fragment), one canamplify a template strand for a given sequence using primers as well asusing ethynyl or azido dNTPs. The prepared DNA fragments can then bepurified and subjected to the click reaction with either azido or alkynedyes or nanoparticles to create a labeled probe. This is suited to bothchromogenically detectable in situ hybridization as well as fluorescent(dyes and Qdots) based probes.

A major advantage that is predicted is that this kind of labeling can bedone at the time of diagnostic or clinical assay. In addition theapplications include automated in situ hybridization platforms such asinstruments from Dako, Ventana Medical Systems, and Vision Biosystemswhere the hybridization can be followed by the Click reaction togenerate the signal.

First strand cDNA synthesis (RT-PCR)

As has been demonstrated, ethynyl-dUTP or azido-dATP are incorporated bythe telomerase enzyme which is a reverse transcriptase (RNA dependentDNA polymerase). Therefore, this methodology can be used to detectproducts of RT-PCR. In this method the nucleotide mix can contain eitheran ethynyl or azido dNTP and enzymes such as reverse transcriptase andDNA polymerase. The product of such an experiment is purified and thensubjected to the Click based labeling method. The final labeled productis purified either by precipitation or size exclusion chromatography.

Second Strand cDNA synthesis (primer extension)

The isothermal DNA extension assay shown above was carried out withvarious different polymerase and serves as an example of second strandcDNA synthesis.

Preparation of RNA probes for FISH

Currently, the two methods of choice for preparation of the RNA FISHprobes are the following:

(1) The small RNA oligonucleotides that can be labeled either viaaminoallyl—NHS ester chemistry.

(2) The incorporation of modified nucleotides using invitrotranscription system to generate a RNA probe.

Based on these two technologies and our understanding of the “Clickable”nucleotides, DNA dependent RNA polymerase from phage T7 or SP6 is usedto incorporate the ethynyl or azido oligonucleotides to produce amodified RNA probe that is subjected to Click chemistry using eitherazido or alkyne fluorescent or chromogenic labels. This is used togenerate fluoregenic or chromogenic RNA probes.

Method to prepare Peptide-nucleic acid Conjugates using Click Chemistry:

A peptide with a O-GlcNac modification on one or more amino acids issubjected to a Gal T1 reaction in the presence of UDP-GalNAz. Thisresults in an azido modified peptide. As explained above, anoligodeoxynucleotide can be created using either alkyne or azido linkednucleotides. A peptide-nucleic acid conjugate is then created byreacting the Azido-linked peptide and ethynyl decorated oligonucleotidesin presence of 1 or 2 mM copper, 10 mM Sodium Ascorbate and 20 mM BCS.

Example 9 Apoptosis Assay

Induce apoptosis in cells using the desired method. It may be desirableto prepare a negative control sample using the cell line of interest byincubating cells in the absence of inducing agent.

Suspend 1-2×10⁶ cells in 0.5 mL of phosphate-buffered saline (PBS). Addthe cell suspension into 5 mL of 1% (w/v) paraformaldehyde in PBS andplace on ice for 15 minutes. Centrifuge the cells for 5 minutes at 300×gand discard the supernatant. Wash the cells in 5 mL of PBS then pelletthe cells by centrifugation. Repeat. Resuspend the cells in 0.5 ml ofPBS. Add the cells to 5 mL of ice-cold 70% (v/v) ethanol. Let the cellsstand for a minimum of 30 minutes on ice or in a -20° C. freezer. Insome biological systems, storage of the cells at −20° C. in 70% (v/v)ethanol for at least 12-18 hours prior to performing the assay yieldsthe best results. Cells can be stored at −20° C. for several days beforeuse.

Resuspend the positive and negative control cells by swirling the vials.Remove 1 mL aliquots of the control cell suspensions (approximately1×10⁶ cells/mL) and place in 12×75 mm flow cytometry centrifuge tubes.Centrifuge (300×g) the control cell suspensions for 5 minutes and removethe 70% (v/v) ethanol by aspiration, being careful to not disturb thecell pellet. Resuspend the control cells of each tube with 1 mL of WashBuffer (Component H of Molecular Probes product A23210). Centrifuge for5 minutes at 300×g and remove the supernatants by aspiration. Repeat.Prepare a DNA-labeling solution; a total volume of 50 μL is required foreach sample. Mix 10 μL of reaction buffer (Compenent G, Molecular ProbesProduct A23210), 0.75 μL of TdT enzyme (terminaldeoxynucleotidyltransferase. Component C, Molecular Probes ProductA23210), 8.0 μL of EdUTP (violet cap) and 31.25 μL of dH₂O. TheDNA-labeling solution is active for approximately 24 hours. Resuspendthe control cell pellets of each tube in 50 μL of the DNA-labelingsolution. Incubate the cells in the DNA-labeling solution for 60 minutesat 37° C. in a temperature controlled bath. Shake the samples every 15minutes to keep the cells in suspension. For samples other than thecontrol cells, incubation times at 37° C. may need to be adjusted tolonger or shorter periods depending on the characteristics of theexperimental samples. The DNA-labeling reaction for the control cellscan also be carried out at 22-24° C. overnight. At the end of theincubation time add 1.0 mL of Rinse Buffer (Compenent I, MolecularProbes product A23210) to each tube and centrifuge at 300×g for 5minutes. Remove the supernatants by aspiration. Repeat the cell rinsingwith 1.0 mL of Rinse Buffer. Centrifuge the samples at 300×g and removethe supernatants by aspiration. Prepare 100 μL of Click Chemistrylabeling solution for each sample by mixing 5.0 μL of the Alexa Fluor488 dye-labeled azide with 95 μL of Rinse Buffer, containing coppersulfate, sodium ascorbate, and BCS in concentrations and proportions asdescribed above. Resuspend the cell pellets in 100 μL of the ClickChemistry labeling solution. Incubate the cells in this solution for 1-3hours at room temperature. Protect the samples from light during theincubation. Add 0.5 mL of the Propidium Iodide/RNase A Staining Buffer(Compenent F, Molecular Probes product A23210) to each sample. Incubatethe cells for an additional 30 minutes at room temperature. Protect thesamples from light during the incubation. Analyze the samples by flowcytometry. It is recommended that the samples be analyzed within 3 hoursof completing the staining procedure. For microscopy applications, it isrecommended that the cells be deposited onto slides after the ClickChemistry labeling staining step, but prior to the propidiumiodide/RNase treatment. Cells that have undergone apoptosis shouldfluorescence brightly when viewed with filter sets appropriate forfluorescein. For adherent cell lines, detached cells present in thesupernatant have a higher probability of being apoptotic than do cellsthat have remained adherent. Detached cells should be collected prior totrypsinization of the adherent cell layer.

Example 10 Digoxin Azide

Mild acid hydrolysis of digoxin will cleave the sugar moieties andprovide the known alcohol derivative. Reaction of this alcohol withphosgene, followed by alkylation with 6-amino-hexanyl-1-azidetrifluoroacetic acid salt, will provide the desired azido-digoxinanalogue.

Example 11 Digoxin Alkyne

Mild acid hydrolysis of digoxin will effectively cleave the sugarmoieties and provide the known alcohol derivative. Reaction of thisalcohol with phosgene, followed by alkylation with propargylamine willprovide the desired alkynyl-digoxin analogue.

Example 12 Synthesis of Dapoxyl® alkyne

The synthesis of Dapoxyl® alkyne is shown in the following reactionscheme.

To a solution of Dapoxyl® carboxylic acid, succinimidyl ester (50 mg,0.12 mmol) in DMF (0.4 mL) at RT was added propargylamine (42 μL, 0.61mmol). The initial clear orange solution turned yellow and cloudy. After˜15 min at RT the reaction was complete, and the solution wasconcentrated to dryness. The residue was purified via HPLC to afford theproduct (36 mg, 84%). TLC (10% EtOAc, CHCl₃) R_(f)=0.30; ESI m/z 346(M⁻, C₂₁H₁₉N₃O₂ requires 346).

Example 13 Synthesis of 5-Carboxytetramethyl Rhodamine Alkyne(5-TAMRA-Alkyne)

The synthesis of 5-Carboxytetramethyl rhodamine alkyne (5-TAMRA-alkyne)is shown in the following reaction scheme.

To a solution of 5-carboxytetramethyl rhodamine, succinimidyl ester(5-TAMRA-SE, 0.10 g, 0.19 mmol) in DMF (0.5 mL) was added propargylamine(25 μL, 0.38 mmol) and H₂O (0.5 mL). After stirring the solution for 30min at RT, the solution was concentrated in vacuo. Purification via HPLC(Phenomenex Prodigy ODS, internal diameter 21.2 mm, eluent 25-40% CH₃CNin 25 mM TEAA, pH 4.7, flow rate of 15 mL/min) gave 68 mg of product(82%, a purple solid) t_(R)=23-33 min. TLC (CH₃CN:H₂O, 8:2) R_(f)=0.67.

Example 14 Synthesis of Biotin Alkyne

The synthesis of Biotin alkyne is shown in the following reactionscheme.

To a solution of EZ-link NHS-PEO₄-biotin (25 mg, 0.004 mmol, Pierce) inDMF (0.1 mL) was added propargylamine (0.1 mL). After stirring thesolution for 90 min at RT, some starting material was still seen.Additional propargylamine (0.2 mL) was added and the solution wasstirred for another 60 min. The solution was concentrated in vacuo. Thecrude material was purified via HPLC to afford 14.4 mg (64%) of theproduct as a yellow solid. TLC (CHCl₃:MeOH, 7:1) R_(f)=0.23; ESI m/z 529(M⁺, C₂₄H₄₀N₄O₇S requires 529).

Example 15 Synthesis of Compound 1

The synthesis of Compound 1 is shown in the following reaction scheme.

To a solution of Alexa Fluor® 488 carboxylic acid, succinimidyl ester,dilithium salt (mixed isomers, 50 mg, 0.079 mmol) in DMF (2.0 mL) wasadded propargylamine (54 □L, 0.79 mmol). The solution was stirredovernight at RT. The initial deep red solution turned pale yellow incolor and became clear. The solution was concentrated in vacuo andpurified via silica gel thin layer chromatography (prep plate, 20% H₂O,CH₃CN) to afford the product (20 mg, 44%) as an orange solid. TLC (3:1,CH₃CN:H₂O) R_(f)=0.70; ESI neg m/z 570 (M⁺, C₂₄H₁₆N₃O₁₀S²⁻ requires570).

Example 16 Synthesis of Compound 2

The synthesis of Compound 2 is shown in the following reaction scheme.

To a solution of Alexa Fluor® 532 carboxylic acid, succinimidyl ester(50 mg, 0.070 mmol) in DMF (2.2 mL) was added propargylamine (100 μL,1.46 mmol). The solution was stirred overnight at RT. H₂O (1.0 mL) wasadded to the solution and the solution was stirred an additional hour.The solution was concentrated in vacuo and the crude material waspurified via HPLC to afford the product (30 mg, 65%). TLC (8:2,CH₃CN:H₂O) R_(f)=0.58; ESI m/z 664 (M⁺, C₃₃H₃₃N₃O₈S₂ requires 664).

Example 17 Synthesis of Compound 3

The synthesis of Compound 3 is shown in the following reaction scheme.

To a solution of Alexa Fluor® 633 carboxylic acid, succinimidyl ester,bis(triethylammonium salt) (50 mg, 0.041 mmol) in DMF (2.0 mL) was addedpropargylamine (28 μL, 0.40 mmol). The solution was stirred overnight atRT. H₂O (1.0 mL) was added to the solution and the solution was stirredan additional hour. The solution was concentrated in vacuo and theproduct (39 mg, 99%). TLC (8:2,CH₃CN:H₂O) R_(f)=0.66; ESI m/z 963 (M⁺,C₄₀H₃₄F₂N₃O₁₁S₆ requires 963).

Example 18 Synthesis of Triarylphosphine-TAMRA Dye for StaudingerLigation

The synthesis of triarylphosphine-TAMRA dye is shown in the reactionscheme below.

To a solution of acid 1(ref: Science 2000, 287, 2007-2010) (80 mg, 0.26mmol) in CH₂Cl₂ (5 mL) was addedN-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDCI, 75mg, 0.39 mmol) and N-hydroxysuccinimide (NHS, 5 mg). The solution wasstirred at RT. After 2.5 h, amine 2 (50 μL, 0.26 mmol) was added and thesolution was stirred overnight. The solution was partitioned betweenCHCl₃ (15 mL) and H₂O (5 mL). The organic layer was separated and theaqueous layer was reextracted with CHCl₃ (15 mL). The combined organiclayers were rinsed once with H₂O (5 mL), followed by saturated aqueousNaCl (5 mL). The organic layer was dried over Na₂SO₄, decanted andconcentrated. The crude was purified via chromatography (silica, 2%MeOH, CHCl₃) to afford the product (99 mg, 71%) as a clear, yellow oil.

To a solution of 3 (10 mg, 0.018 mmol) in CH₂Cl₂ (1.0 mL) was addedtrifluoracetic acid (TFA, 0.5 mL) and the solution was stirred at RT.After 30 min, the solution was concentrated and reevaporated fromtoluene (2×2 mL). The residue (4, 0.018 mmol) was dissolved in DMF (0.2mL) and N-ethyldiisopropylamine (DIEA, 12 □L, 0.72 mmol), and5-carboxytetramethyl rhodamine, succinimidyl ester (5-TAMRA-SE, 9 mg,0.022 mmol) were added. The solution was stirred at RT for 2.5 h,concentrated and purified via silica gel (prep plate, 20% H₂O in CH₃CN)to afford the product (7.4 mg, 48%). TLC (20% H₂O in CH₃CN) R_(f)=0.23;ESI m/z 529 (M⁺, C₂₄H₄₀N₄O₇S requires 529).

Example 19 Synthesis of Triarylphosphine-Biotin for Staudinger Ligation

The synthesis of triarylphosphine-biotin is shown in the followingreaction scheme.

To a solution of 3 (5.3 mg, 0.010 mmol) in CH₂Cl₂ (1.2 mL) was addedtrifluoracetic acid (TFA, 0.5 mL) and the solution was stirred at RT.After 2 h, the solution was concentrated and reevaporated from tolune(2×2 mL). The residue (4, 0.010 mmol) was dissolved in DMF (0.1 mL) andN-ethyldiisopropylamine (DIEA, 3 □L, 0.02 mmol), and EZ-linkNHS-PEO₄-biotin (7 mg, 0.012 mmol) were added. The solution was stirredat RT for 1 h, quenched with saturated NH₄ ⁺Cl⁻ and partitioned betweenCHCl₃ (10 mL) and H₂O (1 mL). The aqueous layer was extracted repeatedlywith CHCl₃ (10 mL per extraction) until no ultraviolet spot was observedby TLC. The combined organic layers were concentrated and purified viasilica gel (prep plate, 7:1 CHCl₃:MeOH) to afford the product (2.2 mg,25%). TLC (7:1 CHCl₃:MeOH, developed 3 times) R_(f)=0.50; ESI m/z 909(M+H⁺, C₄₆H₆₃N₅O₁₀PS requires 909).

Example 20 Synthesis of Cy™5.5Azide

To a solution of 6-(amino)-hexanyl-1-azide trifluoroacetic acid salt(see Scheme 1 for ynthesis, 0.034 mmol) in DMF (0.1 mL) and DIEA (6.0μL, 0.034 mmol) was added Cy™5.5 succinimidyl ester (5 mg, 3.4 nmol).After stirring the solution at RT for 10 min, the reaction solution wasconcentrated in vacuo. The crude was purified via HPLC.

Example 21 Synthesis of Cy™3Azide

To a solution of 6-(amino)-hexanyl-1-azide trifluoroacetic acid salt(see Scheme 1 for synthesis, 0.052 mmol) in DMF (0.1 mL) and DIEA (9.2μL, 0.052 mmol) was added Cy™3 succinimidyl ester (5.0 mg, 5.2 nmol).After stirring the solution at RT for 10 min, the reaction solution wasconcentrated in vacuo. The crude was purified via HPLC.

Example 22 Synthesis of Cy™5.5Alkyne

To a solution of Cy™5.5 succinimidyl ester (GE Amersham, 5.0 mg, 3.7nmol) in DMF (0.1 mL) was added propargylamine (2.5 μL, 0.037 mmol) andH₂O (0.2 mL). The solution was stirred at RT for 30 min thenconcentrated in vacuo. The crude was purified via HPLC.

Example 23 Synthesis of Cy™3Alkyne

To a solution of Cy™3 succinimidyl ester (GE Amersham, 5.0 mg, 5.7 nmol)in DMF (0.1 mL) was added propargylamine (3.9 μL, 0.057 mmol) and H₂O(0.2 mL). The solution was stirred at RT for 30 min then concentrated invacuo. The crude was purified via HPLC.

Example 24 Succinimidyl Ester Azide Synthesis

The synthesis of succinimidyl ester azide is shown in the followingreaction scheme.

6-(Boc-Amino)-hexanyl-1-p-toluenesulfonate

To a solution of 6-(Boc-amino)-1-hexanol (3.0 g, 13.8 mmol) in CHCl₃ (50mL) was added TEA (3.8 mL, 27.6 mmol) and p-toluenesulfonyl chloride(3.9 g, 20.7 mmol). The solution was stirred at RT overnight, dilutedwith CHCl₃ (200 mL), washed with H₂O (4×50 mL), rinsed with brine (1×50mL) and dried over Na₂SO₄. The solution was decanted, concentrated andpurified via silica gel chromatography (6.0×41 cm, 20-70% EtOAc/hexanes)to afford the product as a white solid (3.5 g, 69%). TLC (35%EtOAC/hexanes) R_(f)=0.72, UV active.

6-(Boc-Amino)-hexanyl-1-azide

To a solution of 6-(Boc-amino)-hexanyl-1-p-toluenesulfonate (3.2 g, 8.63mmol) in DMF (21 mL) was added sodium azide (1.12 g, 17.3 mmol). Thesolution was refluxed at 95° C. overnight. After cooling to RT, thesolution was diluted with Et₂O (160 mL) and washed with H₂O (100 mL).The aqueous layer was extracted a second time with Et₂O (100 mL) and thecombined organics were dried over Na₂SO₄. After decanting andconcentrating, the crude material was purified via silica gelchromatography (6×26 cm, 25-30% EtOAc/hexanes) to afford the product asa clear, colorless oil (2.0 g, 97%). TLC, (35% EtOAC/hexanes)R_(f)=0.74, brown spot with ninhydrin stain.

6-(Amino)-hexanyl-1-azide trifluoroacetic acid salt

To a solution of 6-(Boc-amino)-hexanyl-1-azide (0.2 g, 0.83 mmol) inCH₂Cl₂ (1.0 mL) was added TFA (1.0 mL). The solution was stirred at RTfor 2 h, evaporated to dryness and re-evaporated twice from toluene. Theproduct, 6-amino-hexanyl-1-azide trifluoroacetic acid salt (0.83 mmol)was used directly without further purification/

(N-6-Azido-hexanyl)glutaramide

6-Amino-hexanyl-1-azide (0.83 mmol) was dissolved in THF (1.0 mL) andN,N-diisopropylethylamine (0.29 mL, 1.65 mmol) was added. The solutionwas stirred at RT for 10 min then glutaric anhydride (0.47 g, 4.13 mmol)was added. The pale yellow solution was stirred at RT overnight. Thereaction solution was diluted with CHCl₃ (30 mL) and H₂O (10 mL), andacidified to a pH of 1 with 1% HCl; the organic layer was removed. Theaqueous layer was extracted two more times with CHCl₃ (2×30 mL). Thecombined organic layers were rinsed with brine (2×10 mL) and dried overNa₂SO₄. The solution was decanted, and concentrated. The crude waspurified via silica gel chromatography (10% MeOH/CHCl₃ containing 0.1%AcOH) to afford the product as a clear, colorless oil (0.16 g, 75%). Thecolumn was loaded with 10% MeOH/CHCl₃. TLC (10% MeOH/CHCl₃with 0.1%AcOH) R_(f)=0.41, pink with p-anisaldehyde stain, no UV activity.

(N-6-Azido-hexanyl)glutaramide, succinimidyl ester

To a solution of the (N-6-azido-hexanyl) glutaramide (75 mg, 0.29 mmol)in THF (4.0 mL) was added pyridine (110 μL, 1.36 mmol) followed bysuccinimidyl trifluoroacetate (200 mg, 0.95 mmol). The clear, colorlesssolution was stirred at RT for 4 h. The reaction solution was dilutedwith CHCl₃ (20 mL) and rinsed sequentially with 1% AcOH (2×5 mL), H₂O(2×5 mL) and brine (1×5 mL). The crude solution was dried over Na₂SO₄,decanted, and concentrated to afford the product as a clear, colorlessoil (0.10 g, 99%). TLC: (1:1, EtOAc/hexanes) R_(f)=0.64, orange withninhydrin, UV active.

Example 25 Succinimidyl Ester Alkyne Synthesis

The synthesis of succinimidyl ester alkyne is shown in the followingreaction scheme.

10-Undecynoic Acid Succinimidyl Ester

To a solution of 10-undecynoic acid (0.40 g, 2.2 mmol) in CH₃CN (10 mL)was added O-(N-succinimidyl)-N,N,N,N′-tetramethyluroniumtetrafluoroborate (0.99 g, 3.29 mmol). After stirring for 2 min at RT,the reaction was quenched with 1% AcOH and diluted with CHCl₃ (150 mL).The organic solution was then extracted with 1% AcOH (10 mL), rinsedwith H₂O (2×40 mL), then dried over Na₂SO₄. The solution was thendecanted and concentrated. A quantitative yield was assumed and thematerial was taken on directly to the next step. TLC (10% MeOH/CHCl₃)R_(f)=0.90, UV active.

Tent-Butyl Alkyne

To a solution of 10-undecynoic acid succinimidyl ester (0.61 g, 2.19mmol) in CH₃CN (8 mL) was added amino-dPEG™₂-tert-butyl ester (0.46 g,1.97 mmol, Quanta BioDesign) in CH₃CN (2 mL) at RT. After 2 hrs, thesolution was diluted with CHCl₃ (50 mL) and extracted with H₂O (5 mL).The aqueous layer was reextracted with CHCl₃ (2×50 mL). Combinedorganics were dried over Na₂SO₄, decanted and concentrated. The crudewas purified via silica gel chromatography (2.5% MeOH/CHCl₃) to affordthe product as a clear, pale yellow oil (0.48 g, 55%). TLC (9:1CH₃CN:H₂O) R_(f)=0.81.

Succinimidyl Ester Alkyne

To a solution of tert-butyl alkyne (0.48 g, 1.2 mmol) in CH₂Cl₂ (2.0 mL)was added TFA (2.0 mL). The solution was stirred for 1 h, thenconcentrated and reevaporated from toluene (2×1 mL). The resulting brownresidue was dissolved in CH₃CN (5.0 mL) and N,N-diisopropylethylamine(0.84 mL, 4.83 mmol) was added. The solution was stirred at RT for 2min, and then O-(N-succinimidyl)-N,N,N,N′-tetramethyluroniumtetrafluoroborate (0.47 g, 1.56 mmol) was added. After 15 min thereaction was quenched and acidified with 1% AcOH to a pH of 4-5. Thesolution was extracted with CHCl₃ (3×50 mL). The combined organics werereextracted with H₂O (1×10 mL), then dried over Na₂SO₄, decanted andconcentrated to afford a tan solid (0.46 g, 87%). The crude material waspure enough for testing without further purification. TLC (8:2CH₃CN/H₂O) R_(f)=0.79.

Example 26 Iodoacetamide Aazide Synthesis

The synthesis of Iodoacetamide azide is shown in the following reactionscheme.

6-(iodoacetamide)-aminohexanyl-1-azide

To a solution of 6-amino-hexanyl-1-azide trifluoroacetic acid salt (35mg, 0.14 mmol) in DMF (0.1 mL) was added iodoacetic anhydride (0.10 g,0.28 mmol) in the dark. After 2 hr, the reaction was stopped and thesolution was partitioned between CHCl₃ (10 mL) and H₂O (10 mL). Theorganic layer was removed and the aqueous layer was reextracted withCHCl₃ (1×10 mL). The combined organics were rinsed with saturated NaCl(1×5 mL), dried over Na₂SO₄, decanted and concentrated. Purification viasilica gel chromatography (2% MeOH/CHCl₃ containing 0.1% AcOH) providedthe product (35 mg, 81%) as a yellow oil. TLC (10% MeOH/CHCl₃)R_(f)=0.75.

Example 27 Iodoacetamide Alkyne Synthesis

The synthesis of Iodoacetamide alkyne is shown in the following reactionscheme.

N-(iodoacetamide)-propargylamine. To a solution of propargylamine in DMFwas added iodoacetic anhydride in the dark. After 2 hr, the reaction wasstopped and the solution was partitioned between CHCl₃ and H₂O. Theorganic layer was removed and the aqueous layer was reextracted withCHCl₃. The combined organics were rinsed with saturated NaCl, dried overNa₂SO₄, decanted and concentrated.

Example 28 Maleimide Alkyne Synthesis

The synthesis of Maleimide alkyne is shown in the following reactionscheme.

Propargylamine maleimide. After the reaction of propargylamine andmaleic anhydride in the presence of TEA, the intermediate acid wascyclized in the presence of acetic anhydride and sodium acetate at 70°C., to afford the desired propargylamine maleimide.

Example 29 Maleimide Azide Synthesis

The synthesis of Maleimide azide is shown in the following reactionscheme.

N-(6-Azido-aminohexyl)maleimide. After the reaction of6-amino-hexanyl-1-azide trifluoroacetic acid salt and maleic anhydridein the presence of TEA, the intermediate acid was cyclized in thepresence of acetic anhydride and sodium acetate at 70° C., to afford thedesired N-(6-azido-aminohexyl)maleimide.

Example 30 Azido Dyes

5-TAMRA azide. To a solution of 6-(amino)-hexanyl-1-azidetrifluoroacetic acid salt (see Scheme 1 for synthesis, 0.19 mmol) in DMF(0.5 mL) and DIEA (33 μL, 0.19 mmol) was added5-carboxytetramethylrhodamine, succinimidyl ester (5-TAMRA-SE, 50 mg,0.094 mmol). After stirring the solution at RT for 10 min, the reactionsolution was concentrated in vacuo. The crude was purified via silicagel chromatography (prep plate, 9:1 CH₃CN:H₂O) to afford the product asa pink solid (45.6 mg, 87%). TLC (CH₃CN:H₂O, 8:2) R_(f)=0.61, pinkfluorescent spot; ESI-pos m/z 555 M⁺, C₃₁H₃₅N₆O₄ (requires 555).

It is envisioned that any reporter molecule comprising a succinimidylester can be azido modified using the methods described herein. Providedbelow are additional non-limiting examples.

PEG-biotin azide. To a solution of 6-(amino)-hexanyl-1-azidetrifluoroacetic acid salt (see Scheme 1 for synthesis, 0.17 mmol) in DMF(0.5 mL) and DIEA (60 μL, 0.34 mmol) was added NHS-PEO₄-biotin (Pierce,50 mg, 0.08 mmol). After stirring the solution at RT overnight, thesolution was concentrated in vacuo. The crude was purified via silicagel chromatography (7:1 CHCl₃:MeOH) to afford the product as a cloudy,white residue (12.3 mg, 12%). TLC (7:1, CHCl₃:MeOH, 8:2) R_(f)=0.54,faint UV active spot, stains pink with biotin dip; ESI-pos m/z 616 M⁺,C₂₇H₄₉N₇O₇S (requires 616).

Rhodamine Green™ azide (mix of 5- and 6-isomers). To a solution of6-(amino)-hexanyl-1-azide trifluoroacetic acid salt (see Scheme 1 forsynthesis, 0.20 mmol) in DMF (0.5 mL) and DIEA (50 μL, 0.28 mmol) wasadded Rhodamine Green™ carboxylic acid, succinimidyl ester,hydrochloride (mix of 5- and 6-isomers, 50 mg, 0.10 mmol). Afterstirring the solution at RT for 2 h the solution was concentrated invacuo. HPLC (Phenomenex Prodigy ODS, internal diameter 21.2 mm, eluent5-50% CH₃CN (over 60 min) in 25 mM TEAA, pH=4.7, flow rate of 20 mL/min)gave 21.1 mg of product (43%) t_(R)=43-47 min; TLC (CH₃CN:H₂O:AcOH,8:1:1) R_(f)=0.74, fluorescent yellow spot; ESI-pos m/z 499 (M+H,C₂₇H₂₇N₆O₄ requires 499).

Alexa Fluor® 488 azide (5 isomer). To a solution of6-(amino)-hexanyl-1-azide trifluoroacetic acid salt (see Scheme 1 forsynthesis, 0.44 mmol) in DMF (0.5 mL) and DIEA (0.11 mL, 0.88 mmol) wasadded Alexa Fluor® 488 5-carboxylic acid, 2,3,5,6-tetrafluorophenylester, bis(triethylammonium salt) (200 mg, 0.22 mmol). After stirringthe solution at RT for 1 h, the solution was concentrated in vacuo. HPLC(Phenomenex Prodigy ODS, internal diameter 21.2 mm, eluent 0-60% CH₃CN(over 30 min) in 25 mM TEAA, pH=4.7, flow rate of 20 mL/min) gave 58.1mg of product (30%) t_(R)=23-27 min; TLC (CH₃CN:H₂O, 8:2) R_(f)=0.58,fluorescent yellow spot; ESI-neg m/z 657 (M⁻, C₂₇H₂₅N₆O₁₀S₂ ⁻requires657).

Alexa Fuor® 546 azide. To a solution of 6-(amino)-hexanyl-1-azidetrifluoroacetic acid salt (see Scheme 1 for synthesis, 0.093 mmol) inDMF (0.5 mL) and DIEA (32 μL, 0.19 mmol) was added Alexa Fluor® 546carboxylic acid, succinimidyl ester, (50 mg, 0.05 mmol). After stirringthe solution at RT for 2 h, the solution was concentrated in vacuo.

HPLC (Phenomenex Prodigy ODS, internal diameter 21.2 mm, eluent 10-60%CH₃CN (over 60 min) in 25 mM TEAA, pH=4.7, flow rate of 20 mL/min) gave27.2 mg of product (54%) t_(R)=48-52 min; TLC (CH₃CN:H₂O, 9:1)R_(f)=0.24, fluorescent pink spot; ESI-neg m/z 1084 (M⁻,C₄₆H₅₅Cl₃N₇O₁₁S₃ ⁻requires 1084).

Alexa Fluor® 594 azide (5 isomer). To a solution of6-(amino)-hexanyl-1-azide trifluoroacetic acid salt (see Scheme 1 forsynthesis, 0.12 mmol) in DMF (0.5 mL) and DIEA (42 μL, 0.24 mmol) wasadded Alexa Fluor® 594 carboxylic acid, succinimidyl ester *5-isomer*(50 mg, 0.06 mmol). After stirring the solution at RT for 2 h, thesolution was concentrated in vacuo. HPLC (Phenomenex Prodigy ODS,internal diameter 21.2 mm, eluent 25-60% CH₃CN (over 30 min) in 25 mMTEAA, pH=4.7, flow rate of 20 mL/min) gave 16.5 mg of product (32%)t_(R)=23-25 min; TLC (CH₃CN:H₂O, 9:1) R_(f)=0.36, fluorescent red spot;ESI-neg m/z 845 (M⁻, C₄₁H₄₅N₆O₁₀S₂ requires 845).

Dapoxyl azide. To a solution of 6-(amino)-hexanyl-1-azidetrifluoroacetic acid salt (see Scheme 1 for synthesis, 0.25 mmol) in DMF(0.5 mL) and DIEA (43 μL, 0.25 mmol) was added Dapoxyl® carboxylic acid,succinimidyl ester (50 mg, 0.12 mmol). After stirring the solution at RTfor 1 h, the solution was concentrated in vacuo. Purified by SPE(Supelco C18 DSC) to give 41.6 mg of product (78%); ESI-pos m/z 433 (M⁺,C₂₄H₂₈N₆O₂ requires 433).

Alexa Fluor® 568-azide. To a solution of 6-(amino)-hexanyl-1-azide (seeScheme 1 for synthesis, 0.04 mmol) in DMF (0.2 mL) and DIEA (7 82 L,0.04 mmol) was added Alexa Fluor® 568 carboxylic acid, succinimidylester (mix of isomers, 25 mg, 0.02 mmol). After stirring the solution atRT for 2.5 h, H₂O (0.2 mL) was added and the solution was concentratedin vacuo. HPLC (Phenomenex Prodigy ODS, internal diameter 21.2 mm,eluent 20-35% CH₃CN in 25 mM NH₄Ac, pH 4.7, flow rate of 15 mL/min) gave15.3 mg of product (99%) t_(R)=24-30 min; TLC (CH₃CN:H₂O, 8:2)R_(f)=0.63, fluorescent pink spot; ESI-neg m/z 817 (M-2, C₃₉H₄₁N₆O₁₀S₂requires 817).

Example 31 Alkyne Dyes

Oregon Green® 488-alkyne. To a solution of Oregon Green® 488 carboxylicacid, succinimidyl ester (50 mg, 0.98 mmol) in DMF (0.5 mL) was addedpropargylamine (0.26 μL, 0.40 mmol) and H₂O (0.1 mL). After stirring atRT for 15 min, the solution was concentrated. HPLC (Phenomenex ProdigyODS, internal diameter 21.2 mm, eluent 15-30% CH3Cn in 25 mM TEAA pH4.7, flow rate of 15 mL/min) gave 44.5 mg of product (99%) t_(R)=5-13min; TLC (CH₃CN:H₂O, 8:2) R_(f)=0.60, fluorescent yellow spot; ESI-negm/z 448 (M−H⁺, C₂₄H₁₂F₂NO₆ ⁻requires 448).

It is envisioned that any reporter molecule comprising a succinimidylester can be alkyne modified using the methods described herein.Provided below are additional non-limiting examples.

Alkynyl-PEG-biotin. To a solution of NHS-PEO₄-biotin (Pierce, 25 mg,0.004 mmol) in DMF (0.1 mL) at RT was added propargylamine (0.3 mL, 4.5mmol). After stirring for 3 h, the solution was concentrated in vacuoand re-evaporated twice from toluene. HPLC (Phenomenex Prodigy ODS,internal diameter 21.2 mm, eluent 35-50% MeOH in 25 mM NH₄Ac, pH 6.5,flow rate of 15 mL/min) gave 14.4 mg, (64%, a white solid) t_(R)=26-30min; TLC (CHCl₃:MeOH, 7:1) R_(f)=0.20, UV active spot; ESI m/z 529 (M+H⁺, C₂₄H₄₀N₄O₇S requires 529).

5-TAMRA-alkyne. To a solution of 5-carboxytetramethylrhodamine,succinimidyl ester (5-TAMRA-SE, 0.10 g, 0.19 mmol) in DMF (0.5 mL) wasadded propargylamine (25 82 L, 0.38 mmol) and H₂O (0.5 mL). Afterstirring the solution for 30 min at RT, the solution was concentrated invacuo. HPLC (Phenomenex Prodigy ODS, internal diameter 21.2 mm, eluent25-40% CH₃CN in 25 mM TEAA, pH 4.7, flow rate of 15 mL/min) gave 68 mgof product (82%, a purple solid) t_(R)=23-33 min; TLC (CH₃CN:H₂O, 8:2)R_(f)=0.67, fluorescent orange spot; ESI m/z 469 (M+H⁺, C₂₈H₂₆N₃O₄requires 469).

Oregon Green® 488-alkyne. To a solution of Oregon Green® 488 carboxylicacid, succinimidyl ester (50 mg, 0.98 mmol) in DMF (0.5 mL) was addedpropargylamine (0.26 μL, 0.40 mmol) and H₂O (0.1 mL). After stirring atRT for 15 min, the solution was concentrated. HPLC (Phenomenex ProdigyODS, internal diameter 21.2 mm, eluent 15-30% CH3Cn in 25 mM TEAA pH4.7, flow rate of 15 mL/min) gave 44.5 mg of product (99%) t_(R)=5-13min; TLC (CH₃CN:H₂O, 8:2) R_(f)=0.60, fluorescent yellow spot; ESI-negm/z 448 (M−H⁺, C₂₄H₁₂F₂NO₆ ⁻requires 448).

Alexa Fluor® 532-alkyne. To a solution of Alexa Fluor® 532 carboxylicacid, succinimidyl ester (51 mg, 0.07 mmol) in DMF (4.0 mL) was addedpropargylamine (0.1 mL) and H₂O (1.0 mL). The solution was stirred at RTfor 1 h then concentrated in vacuo to afford the crude product. HPLC(Phenomenex Prodigy ODS, internal diameter 21.2 mm, eluent 25-40% CH₃CNin 25 mM NH₄Ac, pH 4.7, flow rate of 15 mL/min) gave 30 mg of product(65%, a red solid) t_(R)=23-30 min; TLC (CH₃CN:H₂O, 1:1) R_(f)=0.58,fluorescent red spot; ESI m/z 664 (M⁺, C₃₃H₃₄N₃O₈S₂ requires 664).

Alexa Fluor® 488-alkyne. To a solution of Alexa Fluor® 488 carboxylicacid, succinimidyl ester, dilithium salt, mixed isomers, (51 mg, 0.08mmol) in DMF (2.0 mL) was added propargylamine (54 μL, 0.80 mmol). Thesolution was stirred at RT for 4 h then concentrated in vacuo. The crudeproduct was purified using column chromatography on silica gel(CH₃CN:H₂O, 8:2) to afford 20 mg (44%, an orange solid). TLC (CH₃CN:H₂O,3:1) R_(f)=0.68; ESI-neg m/z 570 (M-2, C₂₄H₁₆N₃O₁₀S₂ ²⁻requires 570).

Example 32 Triarylphosphine Dye

5-TAMRA-triarylphosphine. To a solution of N-Boc-triarylphosphine-amine(see Scheme 2 for synthesis, 10 mg, 0.018 mmol) in CH₂Cl₂ (1.0 mL) wasadded TFA (0.5 mL). The reaction solution was stirred at RT for 30 min,concentrated in vacuo, and re-evaporated twice from toluene. The crudeamine (0.018 mmol, 99%) was used directly in the next reaction withoutfurther purification.

To a solution of triarylphosphine-amine (0.018 mmol) in DMF (0.2 mL) andDIEA (12 μL, 0.089 mmol) was added 5-carboxytetramethyl rhodamine,succinimidyl ester (5-TAMRA-SE, 9 mg, 0.022 mmol). After stirring thesolution at RT for 2.5 h, the solution was concentrated in vacuo. HPLC(Phenomenex Luna C18(2), internal diameter 10 mm, eluent 40-55% CH₃CN in25 mM NH₄Ac, pH=7, flow rate of 5.0 mL/min) gave 4.1 mg of product (27%)t_(R)=32-34 min; TLC (MeOH:CHCl₃, 1:9) R_(f)=0.67, fluorescent pinkspot; ESI m/z 848 (M+H⁺, C₅₀H₄₈N₄O₇P requires 848).

Example 33

A solid silica glass surface such as a glass slide is derivatized with3-azidopropyl(triethoxy)silane, using standard conditions for covalentattachment of alkyl(trialkoxy)silanes to glass. The residual labelingreagents are rinsed thoroughly, and the azide-derivatized glass isstored under subdued light. The azide functionalized glass surfaces areincubated in water or organic solvent such as methanol with excessacetylene-functionalized partners such as small molecules, dyes,peptides, proteins, enzymes, and nucleic acids over the course of 1-2days in darkness in the presence of excess BCS and Cu(I), which isformed in situ from copper sulfate and sodium ascorbate. The derivatizedglass surface is rinsed thoroughly with water, and stored cold eitherdry or suspended in solution so as to optimize the lifetime of the boundpartner.

Example 34

A solid silica glass surface such as a glass slide is derivatized with3-alkynylpropyl(triethoxy)silane, using standard conditions for covalentattachment of alkyl(trialkoxy)silanes to glass. The residual labelingreagents are rinsed thoroughly, and the alkyne-derivatized glass isstored cold. The alkyne-functionalized glass surfaces are incubated inwater or organic solvent such as methanol with excessazido-functionalized partners such as small molecules, dyes, peptides,proteins, enzymes, and nucleic acids over the course of 1-2 days indarkness in the presence of excess BCS and Cu(I), which is formed insitu from copper sulfate and sodium ascorbate. The derivatized glasssurface is rinsed thoroughly with water, and stored cold either dry orsuspended in solution so as to optimize the lifetime of the boundpartner.

Example 35 Electrophoretic Mobility Shift Assays (EMSA)

This assay is used to determine the specificity and identification ofprotein binding sites on a given ds or ss DNA/RNA molecule. Currentlythe technique uses radiolabeled DNA which is laborious, tedious andrequires high level of safety. With the advent of Click chemistry in DNAlabeling one can perform the same technique with great ease andconvenience.

A ss or ds nucleic acid oligo can be synthesized e.g., by IDT orInvitrogen or Sigma that has a 5′ E-dUMP followed by the sequence ofinterest to the end user. The oligo can then be labeled with and azidoFluorphore and further used for binding to the protein of interestfollowed by PAGE and EMSA. Alternativley the putative sequence of DNA towhich the protein binds can be designed to have a singly azido or alkynenulcoetide. If the protein binds to the sequence it would block accessto the azido dye or tag resulting in no labeling. If the protein doesnot bind to the sequence then the DNA will get readily labeled in theclick reaction giving a clear cut result.

Example 36 Click Labeling by PCR

The reaction was set up as follows:

A 2X SYBR Greener mastermix was used. It was prepared with the samecomponents as the commercial mix (Taq polymerase, buffer, MgCl2, SYBRGreener dye) but without dNTPs and the passive reference dye ROX. Thereaction mixes were prepared according to the following table:

[final] Unmodified dUTP dH2O 6.75 2X mix no ROX 12.5 1X dNTP unmodifiedmix 25 mM 0.2 200 uM each 10 uM B act 300 primers F + R 0.5 200 nM each25 uM ROX 0.05 50 nM 20 Add template 5 Click modified dUTP dH2O 2.95 2Xmix no ROX 12.5 1X Modified dNTP mix 1.2 mM 4 200 uM each 10 uM B act300 primers F + R 0.5 200 nM each 25 uM ROX 0.05 50 nM 20 Add template 5

The template used was Invitrogen qPCR standards, containing 50,000,000(5E7) copies of plasmid with the human B actin CDS, shown below. Thesamples were run as 3 replicates. The location of the primers is shownin FIG. 16, giving a predicted amplicon size of 293 bp.

The cycling conditions were:

Initial denaturation and activation of Taq:

-   95 C 10 minutes-   Amplification:-   1) 95 C 15 seconds-   2) 60 C 30 seconds, fluorescence captured at the end of this step    Repeated for 45 cycles

Melting curve:

The amplicon was heated from 55 C to 95 C in 2 C/minute steps, withfluorescence captured at each step. SBR Green and ROX band pass filterswere used to measure fluorescence, on a MX3000P machine (Stratagene).

Results: The data are shown in FIG. 11 and FIG. 12. The linearamplification plot shown in FIG. 11 shows that the onset of exponentialamplification or threshold cycle (CT) for the click modified dUTP mix isvery similar to that shown for the unmodified dUTP mix. The average CTfor click dUTP is 9.78 vs 10.97 for unmodified dUTP.

The data from the melting curve of FIG. 12 suggests a potentialapplication for Click modified nucleotides as a way to increase the Tmof oligonucleotides. For example, if designing PCR primers in an A-Trich region, this modification could be used to raise the Tm of theprimers so that shorter primers could be made that would show the samemelting temperature as longer primers.

The higher Tm could be used for generating a cDNA library through randompriming. Normally, random 6-8 mers are used. These will naturally have avery low Tm. However the click modified random primers could potentiallybe used at a higher temperature for greater specificity. Researchers areusing octamers to get longer and more specific reads during randompriming but the click modified oligos could provide the enhancedspecificity without the need for longer oligos. Thus, the advantage ofusing short oligos (more matches) can be combined with enhancedhybridization stability.

Example 37 Labeling of Real-Time PCR Products

The product from the real time PCR experiment was either directly orindirectly labeled (PCR product cleaned up using Invitrogen PCRpurification kit prior to Click) with azido-TAMRA in the presence of“Click” conditions. The labeling reaction was composed of a finalconcentration of 25% propylene glycol; 2 mM copper (II); 10 mM BCS, 10mM Sodium Ascorbate and 50 μM azido-TAMRA. The reaction was performedfor 60 minutes at room temperature. This was followed by precipitationof the DNA using 3M sodium acetate, glucagon as a carrier and 100%ethanol. The final DNA pellet was washed twice with 70% ethanol. Thepellet was then dissolved in 50 ul of 10 mM TE buffer pH 8.0. The DNAsolution was warmed up to enhance dissolution of the DNA pellet. 27 ulof the solution was mixed with 3 ul of 10× Blue Juice loading buffer(Invitrogen). 6 ul of the sample was loaded on to a 4-20%TBE—polyacrylamide gel. The gel was run at 10V for 10 minutes; 190V for90 minutes. The gel was pulled out of the cassette and scanned for TAMRA(Ex: 530 nm Em: 580 nm), which is shown as the left image of FIG. 13.The same gel was then stained with SYBR GOLD for 30 minutes and thenscanned as described above, as shown in the right image of FIG. 13.Lanes 1 and 2 are the PCR products that have been generated usingethynyl dUTP and other dNTPs. L is the 25 bp ladder DNA marker. Thearrows point to the ˜300 bp labeled product. FIG. 13 also illustratesthat there is no quenching of the signal.

Example 38 PCR Free Telomerase Assay

TRAPeze™ TELOMERASE assay Chemicon Kit 57700. The experiment issummarized in FIG. 22.

A 250 ul reaction mix (5×) for each of the three different reactionvariables was composed as follows. 25 μl of 10× Trap buffer was mixedwith 50 uM e-dNTPs (50 μM of ethynyl dUTP+50 μM of each of dATP, dGTP,and dCTP) and 344 nM of either the TS primer or biotinylated TS primer.5 μl of Primer Mix (contains three separate primers—a K1 Fwd primer, RPRev Primer and TSK 1 internal control primer from the (TRAPezeTelomerase Kit, Chemicon). The source of telomerase was 1000 positivecontrol cells that are supplied with the Chemicon TRAPeze™ TELOMERASEassay kit. Sau3 cells were used as a negative control as they do notexpress any Telomerase enzyme. Each of the three reactions alsocontained 10 units of Taq DNA polymerase. 1× TRAP reaction buffer (20 mMTris-HCl, pH 8.3, 1.5 MgCl2 63 mM KCl, 0.05% Tween 20, 1 mM EGTA)

The three different reactions were based on

(a) TS primer with +ve control cells (b) Biotinylated TS primer with +vecontrol cells (c) Biotinylated primer with Sau 3 cells.

After the three reaction were set up, ⅕th volume of each of the reactionwas subjected to PCR aided Telomerase assay as has been discussed above.The other ⅘ volumes of each the three reactions were incubated at 300 Cfor 30 minutes and then heated at 950 C for 10 minutes. As shown in theflow chart above, one half of each of the three reactions was cleanedusing size exclusion “Chromaspin” columns. The eluate was then subjectedto click reaction using a final concentration of 25% propylene glycol; 2mM copper (II); 10 mM BCS, 10 mM Sodium Ascorbate and 50 μM azido-TAMRA.The reaction was performed for 30 minutes at room temperature. This wasfollowed by clean up on a size exclusion as described above. The TAMRAlabeled dsDNA (PCR aided Telomerase products) or ss DNA (PCR freeTelomerase products) were blotted on to a Biodyne plus nucleic acidbinding membrane. The membrane was scanned for a TAMRA signal.

The other half of the three reactions were incubated with 25 μl of thestreptavidin coated polystyrene beads (Spherotek) and incubated at 40 Cfor over night. The beads were washed five times with 50 mM Tris pH 8.0.Click chemistry was performed on the oligos attached to the beads usingthe exact same reaction composition and conditions as described above.After incubation, the labeled beads were washed 5 times with 50 mM TrispH 8.0. The beads were semi dried in the speed vac and then scanned forTAMRA.

As seen in FIG. 14, the tube containing beads coated with biotinylatedTS primer show a signal for TAMRA, while the tubes with either TS primeror negative control cells do not show the signal.

Additionally, as shown in FIG. 15, spots on the PCR aided +ve controland TS biotinylated primer show TAMRA signal on the Biodyne nucleic acidmembrane. The signal intensity from the PCR positive control is fargreater than PCR free Telomerase assay.

Example 39 Tagging Phosphoproteins Using Nucleotide Analogs

Phosphoproteins are labeled in vivo or in vitro using alkyne orazide-tagged nucleotides whereby the azide or alkyne moiety is placed onthe gamma phosphate. For example one of the nucleotides shown below isadded to a reaction mixture containing a protein kinase and a kinasetarget molecule. After tagging the molecule is reacted with theappropriate alkyne or azide detection or affinity reagent forquantitation, visualization, or enrichment. In one example reaction,modified nucleotide substrates may be added directly to cultured cellsfor metabolic incorporation of the tagged gamma-phosphate molecule intocellular macromolecules including proteins. The process may involvetreatment of the cells with pharmacological agents to detect alterationsin phosphorylation dynamics. Entry of the compounds into live culturedcells could be enhanced by modifying the nucleotides with functionalgroups that would afford permeability, or by concomitant addition ofcell permeablizing agents. In another example reaction, the kinasereaction could be performed in vitro using cellular extracts as thesource of kinases and substrates. The modified nucleotides is added tothe reaction mixture and the reaction mixtures incubated with or withoutthe addition of pharmacological agents of interest. The in vitroreaction optionally entails adding an exogenous kinase or substratesource to the cellular extract along with the nucleotide analogs. Inanother application, the method is used in vitro without cellularextracts, using purified kinases and kinase substrates. In all of thedisclosed examples the reaction mix may contain a buffer optimized forthe particular kinases of interest, a kinase source, a metal ion source,glycerol, nucleotide ATP analog, and ATP. The “click” detection reactionwith an alkyne probe would be performed in the presence of copper(I), orcopper(II) in the presence of a copper(II) reducing agent, a copper(I)chelating agent, and an appropriate buffer to maintaing optimal pHconditions.

The reagents employed in the examples are commercially available or canbe prepared using commercially available instrumentation, methods, orreagents known in the art. The foregoing examples illustrate variousaspects of the invention and practice of the methods of the invention.The examples are not intended to provide an exhaustive description ofthe many different embodiments of the invention. Thus, although theforgoing invention has been described in some detail by way ofillustration and example for purposes of clarity of understanding, thoseof ordinary skill in the art will realize readily that many changes andmodifications can be made thereto without departing from the spirit orscope of the appended claims.

Each of the references cited herein are hereby incorporated by referenceas if set forth fully herein.

1. A method of forming a nucleic acid conjugate, wherein the method comprises: a) incorporating an azide modified nucleotide into the nucleic acid polymer by contacing the azide modified nucleotide nucleotide with at least one other nucleotide in the presence of a DNA amplification enzyme to form an azide modified nucleic acid polymer; and b) contacting the azide modified nucleic acid polymer with a reporter molecule, carrier molecule or solid support that comprises an activated or terminal alkyne or phosphine moiety to form a nucleic acid polymer-reporter molecule, carrier molecule, solid support conjugate.
 2. A method of forming a nucleic acid conjugate, wherein the method comprises: a) incorporating a terminal alkyne modified nucleotide into the nucleic acid polymer by contacing the terminal alkyne modified nucleotide nucleotide with at least one other nucleotide in the presence of a DNA amplification enzyme to form a terminal alkyne modified nucleic acid polymer; and b) contacting the terminal alkyne modified nucleic acid polymer with a reporter molecule, carrier molecule or solid support that comprises an azido moiety to form a nucleic acid polymer-reporter molecule, carrier molecule, solid support conjugate.
 3. A method of forming a nucleic acid conjugate, wherein the method comprises: a) incorporating a phosphine modified nucleotide into the nucleic acid polymer by contacing the phosphine modified nucleotide nucleotide with at least one other nucleotide in the presence of a DNA amplification enzyme to form a phosphine modified nucleic acid polymer; and b) contacting the phosphine modified nucleic acid polymer with a reporter molecule, carrier molecule or solid support that comprises an azido moiety to form a nucleic acid polymer-reporter molecule, carrier molecule, solid support conjugate.
 4. A method for making an azido, alkyne or phosphine modified nucleic acid polymer, wherein the method comprises: incubating at least one azido, alkyne or phosphine modified nucleotide in the presence of a nucleic acid amplification enzyme to form an azido, alkyne or phosphine modified nucleic acid polymer.
 5. The method according to claim 4, wherein the nucleic acid enzyme is a DNA polymerase.
 6. The method according to claim 4, wherein the nucleic acid enzyme is a RNA polymerase.
 7. The method according to claim 4, wherein the melting temperature of the azido, alkyne or phosphine modified nucleic acid polymer is increased.
 8. The method according to claim 1, wherein the reporter molecule is a xanthene, cyanine, coumarin, borapolyazaindacene or pyrene dye.
 9. The method according to claim 1, wherein the reporter molecule is an enzyme substrate or hapten.
 10. The method according to claim 1, wherein the carrier molecule is an amino acid, a peptide, a protein, a polysaccharide, a nucleotide, a nucleoside, an oligonucleotide, a nucleic acid, a hapten, a psoralen, a drug, a hormone, a lipid, a lipid assembly, a synthetic polymer, a polymeric microparticle, a biological cell or a virus.
 11. The method according to claim 1, wherein the carrier molecule comprises an antibody or fragment thereof, an avidin or streptavidin, a biotin, a blood component protein, a dextran, an enzyme, an enzyme inhibitor, a hormone, an IgG binding protein, a fluorescent protein, a growth factor, a lectin, a lipopolysaccharide, a microorganism, a metal binding protein, a metal chelating moiety, a non-biological microparticle, a peptide toxin, a phosphotidylserine-binding protein, a structural protein, a small-molecule drug, or a tyramide.
 12. The method according to claim 1, wherein the solid support is a microfluidic chip, a silicon chip, a microscope slide, a microplate well, silica gels, polymeric membranes, particles, derivatized plastic films, glass beads, cotton, plastic beads, alumina gels, polysaccharides, polyvinylchloride, polypropylene, polyethylene, nylon, latex bead, magnetic bead, paramagnetic bead, or superparamagnetic bead.
 13. The method according to claim 1, wherein the solid support is Sepharose, poly(acrylate), polystyrene, poly(acrylamide), polyol, agarose, agar, cellulose, dextran, starch, FICOLL, heparin, glycogen, amylopectin, mannan, inulin, nitrocellulose, diazocellulose or starch.
 14. A method of detecting an azido modified nucleic acid polymer, comprising: a) forming an azide-alkyne cycloaddition reaction mixture comprising: a reporter molecule that comprises a terminal alkyne moiety: an azido modified nucleic acid polymer; b) incubating the azide-alkyne cycloaddition reaction mixture for a sufficient amount of time to form a nucleic acid polymer-reporter molecule conjugate; c) separating the nucleic acid polymer-reporter conjugate by size and/or weight of the nucleic acid polymer-reporter-reporter molecule conjugate to form a separated nucleic acid polymer-reporter-reporter molecule conjugate; d) illuminating the separated nucleic acid polymer-reporter-reporter molecule conjugate with an appropriate wavelength to form an illuminated nucleic acid polymer-reporter- reporter molecule conjugate; e) observing the illuminated nucleic acid polymer-reporter-reporter molecule conjugate wherein the nucleic acid polymer is detected.
 16. The method according to claim 14, wherein step a) further comprises a. copper ions; b. at least one reducing agent; and c. a copper chelator.
 16. The method according to claim 14, wherein the reporter molecule is xanthene, cyanine, coumarin, borapolyazaindacene or pyrene dye.
 17. The method according to claim 14, wherein the reporter molecule is an enzyme substrate, fluorescent protein or hapten.
 18. The method according to claim 15, wherein the copper chelator is a copper (I)chelator.
 19. The method according to claim 15, wherein the copper chelator is N,N,N′,N′-tetrakis(2-pyridylmethyl)ethylenediamine (TPEN), EDTA, neocuproine, N-(2-acetamido)iminodiacetic acid (ADA), pyridine-2,6-dicarboxylic acid (PDA), S-carboxymethyl-L-cysteine (SCMC), 1,10 phenanthroline, or a derivative thereof, trientine, glutathione, histadine, polyhistadine or tetra- ehylenepolyamine (TEPA).
 20. The method according to claim 15, wherein the copper chelator is 1,10 phenanthroline, bathophenanthroline disulfonic acid (4,7odiphenyl-1,10-phenanthroline disulfonic acid) or bathocuproine disulfonic acid (2,9-dimethyl-4,7-diphenyl-1,10-phenanthroline disulfonate).
 21. The method according to claim 15, wherein the reducing agent is acorbate, Tris(2-Carboxyethyl) Phosphine (TCEP), TCP (2,4,6-trichlorophenol), NADH, NADPH, thiosulfate, 2-mercaptoethanol, dithiothreotol, glutathione, cysteine, metallic copper, quinone, hydroquinone, vitamin K₁, Fe²⁺, Co²⁺, or an applied electric potential.
 22. The method according to claim 15, wherein the reducing agent is ascorbate.
 23. The method according to claim 14, wherein the separating step comprises chromatography or electrophoresis.
 24. The method according to claim 23, wherein the chromatography comprises one or more of FPLC, HPLC, liquid chromatograpy (LC), size exclusion chromatography, ion exchange chromatography, or affinity chromatography.
 25. The method according to claim 23, wherein electrophoresis comprises gel electrophoresis, 1 dimensional (1D) gel electrophoresis, 2 dimensional (2D) gel electrophoresis, native gel electrophoresis, denaturing gel electrophoresis, isoelctric focusing, or capillary electrophoresis.
 26. An azide-alkyne cycloaddition reaction mixture comprising: a reporter molecule that comprises a terminal alkyne moiety: an azido modified nucleic acid; copper ions; at least one reducing agent; and a copper chelator.
 27. A method for detecting immobilized azido modified nucleic acids, wherein the method comprises: a) immobilizing the azido modified nucleic acids on a solid or semi-solid matrix to form an immbolized azido modified nucleic acid; b) contacting the immbolized azido modified nucleic acid with a reporter molecule that contains an azide reactive group to form a contacted azido modified nucleic acid; c) incubating the contacted azido modified nucleic acid for a sufficient amount of time to form a reporter molecule-nucleic acid conjugate; d) illuminating the reporter molecule-nucleic acid conjugate with an appropriate wavelength to form an illuminated reporter molecule-nucleic acid conjugate; e) observing the illuminated reporter molecule-nucleic acid conjugate whereby the immobilized azido modified nucleic acid is detected.
 28. A method for detecting immobilized alkyne modified nucleic acids, wherein the method comprises: a) immobilizing the alkyne modified nucleic acids on a solid or semi-solid matrix to form an immbolized alkyne modified nucleic acid; f) contacting the immbolized alkyne modified nucleic acid with a reporter molecule that contains an azido group to form a contacted alkyne modified nucleic acid; g) incubating the contacted alkyne modified nucleic acid for a sufficient amount of time to form a reporter molecule-nucleic acid conjugate; h) illuminating the reporter molecule-nucleic acid conjugate with an appropriate wavelength to form an illuminated reporter molecule-nucleic acid conjugate; i) observing the illuminated reporter molecule-nucleic acid conjugate whereby the immobilized alkyne modified nucleic acid is detected.
 29. The method according to claim 28, wherein the solid or semi-solid support is a slide, an array, an agarose gel, a polyacrylamide gel, a hydrogel, a polymeric particle or glass.
 30. A kit comprising: Azide or alkyne-dUTP; a telomerase enzyme; an azide or alkyne reactive reporter molecule, carrier molecule or solid support.
 31. A kit for labeling a nucleic acid polymer comprising: at least one nucleotide analogue that comprises an azide, alkyne or phosphine moiety; and a reporter molecule, carrier molecule or solid support comprising an azide, alkyne or phosphine moiety.
 32. The kit according to claim 31, further comprising a nucleici acid amplification enzyme.
 33. A method of measuring Telomerase Enzyme Activity, comprising steps of: a) contacting a cell with an effective amount of a dNTP nucleotide that comprises an azide group and a Telomerase enzyme such that the dNTP nucleotide is incorporated into at least one nucleic acid polymer; b) contacting the nucleic acid polymer with a reporter molecule comprising an alkyne or phosphine moiety to form a azido modified nucleic acid polymer reporter molecule conjugate; c) separating the azido modified nucleic acid polymer reporter molecule conjugate from nucleic acid polymers that do not comprise a reporter molecule, and d) illuminating the azido modified nucleic acid polymer reporter molecule conjugate to determine Telomerase activity.
 34. A method of measuring Telomerase Enzyme Activity, comprising steps of: a) contacting a cell with an effective amount of a dNTP nucleotide that comprises an alkyne or phosphine group and a Telomerase enzyme such that the dNTP nucleotide is incorporated into at least one nucleic acid polymer; b) contacting the nucleic acid polymer with a reporter molecule comprising an azido moiety to form an alkyne or phosphine modified nucleic acid polymer reporter molecule conjugate; c) separating the alkyne or phosphine modified nucleic acid polymer reporter molecule conjugate from nucleic acid polymers that do not comprise a reporter molecule, and d) illuminating the alkyne or phosphine modified nucleic acid polymer reporter molecule conjugate to determine Telomerase activity. 